Amino acid sequences directed against her2 and polypeptides comprising the same for the treatment of cancers and/or tumors

ABSTRACT

The present invention relates to amino acid sequences and Nanobodies that are directed against Epidermal Growth Factor Receptor 2 (HER2), as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences.

The present invention relates to amino acid sequences that are directedagainst (as defined herein) Epidermal Growth Factor Receptor 2 (HER2),as well as to compounds or constructs, and in particular proteins andpolypeptides, that comprise or essentially consist of one or more suchamino acid sequences (also referred to herein as “amino acid sequencesof the invention”, “compounds of the invention”, and “polypeptides ofthe invention”, respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

HER2 (also known as HER-2, Her-2, ErbB-2, ERBB2, EGF receptor 2,HER2/neu) is a member of the ErbB protein family, also known as the ERBBor the epidermal growth factor receptor family. This subclass I of thereceptor tyrosine kinase (RTK) superfamily comprises four members:EGFR/ERBB1, HER2/ERBB2, HER3/ERBB3 and HER4/ERBB4. All members have anextracellular ligand-binding region, a single membrane-spanning regionand a cytoplasmic tyrosine-kinase-containing domain. The ERBB receptorsare expressed in various tissues of epithelial, mesenchymal and neuronalorigin. Under normal physiological conditions, activation of the ERBBreceptors is controlled by the spatial and temporal expression of theirligands, which are members of the EGF family of growth factors (Rieseand Stern, 1998, Bioessays 20: 41; Yarden and Sliwkowski, 2001, NatureRev. Mol. Cell. Biol. 2: 127). Ligand binding to ERBB receptors inducesthe formation of receptor homo- and heterodimers and activation of theintrinsic kinase domain, resulting in phosphorylation on specifictyrosine residues within the cytoplasmic tail. These phosphorylatedresidues serve as docking sites for a range of proteins, the recruitmentof which leads to the activation of intracellular signalling pathways(Yarden and Sliwkowski, 2001, Nature Rev. Mol. Cell. Biol. 2: 127;Olayioye et al. 2000, EMBO J. 19: 3159; Schlessinger, 2004, Science 306:1506; Hynes and Lane, 2005, Nature Reviews/Cancer 5: 341).

For the amino acid sequence of HER-2, reference is made to the sequencesmentioned under Genbank accession numbers NM 001005862 en NM 004448(both incorporated herein by reference). For the domain(s) of HER-2involved in the interaction between HER-2 and Omnitarg and the aminoacid sequence(s) thereof, reference is made to Franklin et al. (2004,Cancer cell 5:317-328; also incorporated herein by reference). For thedomains of HER-2 involved in the interaction between HER-2 andHerceptin® and the amino acid sequence(s) thereof, reference is made toCho et al. (2003, Nature 421:756-760; also incorporated herein byreference).

HER2 is thought to be an orphan receptor, with none of the EGF family ofligands able to activate it. However, ErbB receptors dimerise on ligandbinding, and HER2 is the preferential dimerisation partner of othermembers of the ErbB family (Graus-Porta et al, 1997, EMBO J. 16: 1647).

The extracellular region of each ERBB receptor consists of four domains(I-IV). The structure of HER2's extracellular region is radicallydifferent from the other EGF receptors. In the other EGF receptors, innon-activated state, domain II binds to domain IV. Upon binding todomains I and III, the activating growth factor (ligand) selects andstabilizes a conformation that allows a dimerization arm to extend fromdomain II to interact with an ERBB dimer partner. HER2, on the otherhand, has a fixed conformation that resembles the ligand-activatedstate: the domain II-IV interaction is absent and the dimerization loopin domain II is continuously exposed (in detail discussed in Hynes andLane, 2005, Nature Reviews/Cancer 5: 341, Garrett et al. 2003, Mol.Cell. 11: 495; Cho et al. 2003, Nature 421: 756). This also explains whyHER2 is the preferred dimerization partner.

Amplification of HER-2 leading to overexpression of the receptor,originally detected in a subset of breast tumours, occurs in varioushuman cancers including ovarian, stomach, bladder, gastric and salivarycancers (Holbro and Hynes, 2004, Annu. Rev. Pharmacol. Toxicol. 44:195;Hynes and Stern, 1994, Biochim. Biophys. Acta 1198: 165). Approximately25-30 percent of breast cancers have an amplification of the HER2/neugene or overexpression of its protein product. Overexpression of thisreceptor in breast cancer is associated with increased diseaserecurrence and worse prognosis. Therefore, ERBB receptors have beenintensely pursued as therapeutic targets (Holbro and Hynes, 2004, Annu.Rev. Pharmacol. Toxicol. 44:195).

mAb4D5, isolated by Ullrich et al. (Mol. Cell. Biol. 1989, 9: 1165), andtrastuzumab (marketed as Herceptin®), its humanized (human IgG1backbone, murine complementary-determining regions) variant (Carter etal. 1992, Proc. Natl. Acad. Sci. USA 89: 4285), block proliferation ofHER2-overexpressing breast cancer cells. The structure of thetrastuzumab Fab fragment bound to the extracellular portion of HER2indicates that its epitope is toward the carboxyterminus of domain IV(Cho et al. 2003, Nature 421: 756). Domain IV does not participate inreceptor dimerization, and blockade of dimerization does not explain themechanism of action of this antibody. The mechanisms underlyingtrastuzumab's clinical efficacy is still under debate and seems to bemultifaceted. Its inherent ability to recruit immune effector cells suchas macrophages and monocytes to the tumor through the binding of itsconstant Fc domain to specific receptors on these cells, might berelevant for its anti-tumor activity. In addition to this Fc-mediatedfunctions, preclinical studies have shown that the antibodydownregulates HER2 levels (Hudziak et al. 1989, Mol. Cell. Biol 9: 1165)and HER2-mediated signaling pathways (Lane et al. 2000, Mol. Cell. Biol.20: 3210, Motoyama et al. 2002, Cancer Res. 62: 3151). Furthermore,metalloproteinase-mediated. HER2 ectodomain shedding has been proposedto cause constitutive HER2 signaling and trastuzumab also blocks thisprocess (Molina et al. 2001, Cancer Res. 61: 4744). Trastuzumab is onlyeffective in breast cancer where the HER2/neu receptor is overexpressed.Clinical trials showed that the addition of trastuzumab to standardchemotherapy prolonged relapse-free survival, leading to the approval ofthe drug for treatment of HER2-overexpressing metastatic breast cancerpatients.

Another monoclonal antibody, pertuzumab (Omnitarg) (Olayioye, 2001,Breast Cancer Res 3: 385), which inhibits ligand activation of an ErbBhetero-oligomer comprising HER2 and HER3, HER4 or EGFR, is in advancedclinical trials. Pertuzumab binds to HER2 near the center of domain II.Binding is predicted to sterically block the region necessary for HER2dimerization with other ERBBs (Franklin et al. 2004, Cancer Cell 5:317), Pertuzumab but not trastuzumab inhibits the growth of tumorsdisplaying low HER2 levels (Agus et al. 2002, Cancer Cell 2: 127).

A specific, but non-limiting object of the invention is to providetherapeutic compounds that have improved therapeutic and/orpharmacological properties and/or other advantageous properties (suchas, for example, improved ease of preparation and/or reduced costs ofgoods), compared to these conventional antibodies. These improved andadvantageous properties will become clear from the further descriptionherein. The therapeutic compounds provided by the invention may, forexample, have an increased avidity and/or potency, an increasedselectivity and/or they may be capable of partially or totally blockingcertain (one or more) sites.

The polypeptides and compositions of the present invention can generallybe used to bind HER2 and, by this binding to HER2, modulate, and inparticular inhibit or prevent, the signalling that is mediated by HER2,to modulate the biological pathways in which HER2 is involved, and/or tomodulate the biological mechanisms, responses and effects associatedwith such signalling or these pathways (which are also referred toherein as “modes of action” of the polypeptides and compositions of theinvention).

One specific, non-limiting, object of the invention is to providetherapeutic compounds that combine two or more modes of action, e.g. byblocking of two or more different cell signalling pathways. Onespecific, but non-limiting object of the invention is to providetherapeutic compounds that combine the mode of action of Herceptin® andOmnitarg.

The polypeptides and compositions of the present invention can be usedto modulate, and in particular inhibit and/or prevent, dimerization ofHER2 with an ERBB receptor, and thus to modulate, and in particularinhibit or prevent, the signalling that is mediated by dimerization ofHER2 with said ERBB receptor, to modulate the biological pathways inwhich HER2 and/or said ERBB receptor are involved, and/or to modulatethe biological mechanisms, responses and effects associated with suchsignalling or these pathways.

As such, the polypeptides and compositions of the present invention canbe used for the prevention and treatment (as defined herein) of cancersand/or tumors. Generally, “cancers and/or tumors” can be defined asdiseases and disorders that can be prevented and/or treated,respectively, by suitably administering to a subject in need thereof(i.e. having the disease or disorder or at least one symptom thereofand/or at risk of attracting or developing the disease or disorder) ofeither a polypeptide or composition of the invention (and in particular,of a pharmaceutically active amount thereof) and/or of a known activeprinciple active against HER2 or a biological pathway or mechanism inwhich HER2 is involved (and in particular, of a pharmaceutically activeamount thereof). Examples of such cancers and/or tumors will be clear tothe skilled person based on the disclosure herein, and for exampleinclude the following diseases and disorders: breast cancer and/ortumors, ovarian cancer and/or tumors, stomach cancer and/or tumors,bladder cancer and/or tumors, gastric cancer and/or tumors, salivarycancer and/or tumors, and prostate cancer.

In particular, the polypeptides and compositions of the presentinvention can be used for the prevention and treatment of cancers and/ortumors which are characterized by excessive and/or unwanted signallingmediated by HER2 or by the pathway(s) in which HER2 is involved.Examples of such cancers and/or tumors will again be clear to theskilled person based on the disclosure herein.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate HER2-mediatedsignalling, such as those mentioned in the prior art cited above. It isalso envisaged that the polypeptides of the invention can be used toprevent and/or to treat all diseases and disorders for which treatmentwith such active principles is currently being developed, has beenproposed, or will be proposed or developed in future. In addition, it isenvisaged that, because of their favourable properties as furtherdescribed herein, the polypeptides of the present invention may be usedfor the prevention and treatment of other diseases and disorders thanthose for which these known active principles are being used or will beproposed or developed; and/or that the polypeptides of the presentinvention may provide new methods and regimens for treating the diseasesand disorders described herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of cancers and/ortumors and of the further diseases and disorders mentioned herein; andto provide methods for the diagnosis, prevention and/or treatment ofsuch diseases and disorders that involve the administration and/or useof such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of cancers and/or tumors and of the furtherdiseases and disorders mentioned herein; and to provide methods for thediagnosis, prevention and/or treatment of such diseases and disordersthat involve the administration and/or the use of such therapeuticproteins and compositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against (as defined herein) HER2,in particular against HER2 from a warm-blooded animal, more inparticular against HER2 from a mammal, and especially against human HER2(and specifically, against human HER-2 with the amino acid sequencegiven under Genbank accession numbers NM 001005862 en NM 004448); and toprovide proteins and polypeptides comprising or essentially consistingof at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith HER2 and/or mediated by HER2 (such as the diseases, disorders andconditions mentioned herein) in a warm-blooded animal, in particular ina mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by HER2 (such as thediseases, disorders and conditions mentioned herein) in a warm-bloodedanimal, in particular in a mammal, and more in particular in a humanbeing.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to HER2; as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence.

More in particular, the invention provides amino acid sequences that canbind to HER2 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,        more preferably between 10⁴M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as        between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to HER2 with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to HER2 will become clear from the furtherdescription and examples herein.

For binding to HER2, an amino acid sequence of the invention willusually contain within its amino acid sequence one or more amino acidresidues or one or more stretches of amino acid residues (i.e. with each“stretch” comprising two or more amino acid residues that are adjacentto each other or in close proximity to each other, i.e. in the primaryor tertiary structure of the amino acid sequence) via which the aminoacid sequence of the invention can bind to HER2, which amino acidresidues or stretches of amino acid residues thus form the “site” forbinding to HER2 (also referred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than HER2), so as to provide a monovalent,multivalent or multispecific polypeptide of the invention, respectively,all as described herein. Such a protein or polypeptide may also be inessentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that Nanobodies—as describedherein—may sometimes contain a disulphide bridge between CDR3 and CDR1or FR2). However, it should be noted that one or more amino acidsequences of the invention may be linked to each other and/or to otheramino acid sequences (e.g. via disulphide bridges) to provide peptideconstructs that may also be useful in the invention (for example Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs. Reference is for example made to the review byHolliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human HER2; whereas for veterinary purposes, the amino acidsequences and polypeptides of the invention are preferably directedagainst HER2 from the species to be treated, or at least cross-reactivewith HER2 from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against HER2,contain one or more further binding sites for binding against otherantigens, proteins or targets. The efficacy of the amino acid sequencesand polypeptides of the invention, and of compositions comprising thesame, can be tested using any suitable in vitro assay, cell-based assay,in vivo assay and/or animal model known per se, or any combinationthereof, depending on the specific disease or disorder involved.Suitable assays and animal models will be clear to the skilled person,and for example include BIAcore binding assay, FACS binding and/orcompetition assay, ELISA binding and/or competition assay, FMAT bindingand/or competition assay, Alphascreen binding and/or competition assay,tumor (e.g. SKBR3) cell proliferation assay (Hudziak et al., Molecularand Cellular Biology 9:1165-1172, 1989), cell signalling assays (Agus etal., Cancer Cell 2:127-136, 2002), SCID mice with implanted tumor (i.e.Xenograft mice) (Agus et al., Cancer Cell 2:127-136, 2002),HER2-transgenic mice (Scwall et al., Breast Cancer Res 5(Suppl 1):14,2003), as well as the assays and animal models used in the experimentalpart below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against HER2 from a first species of warm-bloodedanimal may or may not show cross-reactivity with HER2 from one or moreother species of warm-blooded animal. For example, amino acid sequencesand polypeptides directed against human HER2 may or may not show crossreactivity with HER2 from one or more other species of primates (suchas, without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomologus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with HER2from one or more species of animals that are often used in animal modelsfor diseases (for example mouse, rat, rabbit, pig or dog), and inparticular in animal models for diseases and disorders associated withHER2 (such as the species and animal models mentioned herein). In thisrespect, it will be clear to the skilled person that suchcross-reactivity, when present, may have advantages from a drugdevelopment point of view, since it allows the amino acid sequences andpolypeptides against human. HER2 to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with HER2 from multiple species of mammal willusually be advantageous for use in veterinary applications, since itwill allow the same amino acid sequence or polypeptide to be used acrossmultiple species. Thus, it is also encompassed within the scope of theinvention that amino acid sequences and polypeptides directed againstHER2 from one species of animal (such as amino acid sequences andpolypeptides against human HER2) can be used in the treatment of anotherspecies of animal, as long as the use of the amino acid sequences and/orpolypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain (I, II, III and/or IV), subunit or conformation (whereapplicable) of HER2 against which the amino acid sequences andpolypeptides of the invention are directed. For example, the amino acidsequences and polypeptides may or may not be directed against an“interaction site” (as defined herein). However, it is generally assumedand preferred that the amino acid sequences and polypeptides of theinvention are preferably at least directed against an interaction site(as defined herein), and in particular against the Herceptin® bindingsite on HER2 (see Cho et al, (2003), Nature 421:756-760), the Omnitargbinding site on HER2 (see Franklin et al. (2004), Cancer cell5:317-328), or the Herceptin® binding site and the Omnitarg binding siteon HER2.

An amino acid of the invention that is directed against and/or binds onespecific antigenic determinant, or epitope of a target or antigen (suchas a specific antigenic determinant, epitope, part, domain (I, II, IIIand/or IV) or subunit of HER2) while not binding any other antigenicdeterminant, or epitope of the target or antigen and not binding anyother target or antigen, is also referred to herein as monovalent aminoacid or monovalent construct of the invention.

As further described herein, a polypeptide of the invention may containtwo or more (monovalent) amino acid sequences or monovalent constructsof the invention that are directed against HER2. Generally, suchpolypeptides will bind to HER2 with increased avidity compared to asingle amino acid sequence of the invention. Such a polypeptide may forexample comprise two amino acid sequences of the invention that aredirected against the same antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of HER2 (which may or may notbe an interaction site); or comprise at least one “first” amino acidsequence of the invention that is directed against a first sameantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) of HER2 (which may or may not be an interactionsite); and at least one “second” amino acid sequence of the inventionthat is directed against a second antigenic determinant, epitope, part,domain, subunit or confirmation (where applicable) different from thefirst (and which again may or may not be an interaction site).Preferably, in such “biparatopic” polypeptides of the invention, atleast one amino acid sequence of the invention is directed against aninteraction site (as defined herein), although the invention in itsbroadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the amino acid sequences and polypeptidesmay be such that they compete with the cognate binding partner (e.g. theligand, receptor or other binding partner, as applicable) for binding tothe target, and/or such that they (fully or partially) neutralizebinding of the binding partner to the target.

Thus, in one preferred, but non-limiting aspect, the amino acidsequences and polypeptides of the invention are directed against theHerceptin® binding site on HER2 and/or are capable of competing withHerceptin® for binding to HER-2, as determined using a suitablecompetition assay, such as the assay described in. Example 8. Such aminoacid sequences and polypeptides of the invention may be as furtherdefined herein. The amino acid sequences and polypeptides of theinvention may in particular be directed against domain IV of HER2. In apreferred aspect, the amino acid sequences and polypeptides of theinvention are directed against the C-terminus of domain IV of HER2.

In another preferred, but non-limiting aspect, the amino acid sequencesand polypeptides of the invention are capable, upon binding to HER-2, to(i) recruit immune effector cells such as macrophages and monocytes tothe tumor (for this purpose, most preferably a polypeptide of theinvention is used that contains an Fc portion that confers upon thepolypeptide the ability to recruit immune effector cells such asmacrophages and monocytes to the tumor); and/or (ii) modulate (asdefined herein) HER-2 or HER-2 mediated signalling by downregulatingHER2 levels (for example, as determined by the methodology described inHudziak et al. 1989, Mol. Cell. Biol 9: 1165) and/or by downregulatingHER2-mediated signaling pathways (for example, as determined by themethodology described in Lane et al. 2000, Mol. Cell. Biol. 20: 3210,Motoyama et al. 2002, Cancer Res. 62: 3151); and/or (iii) modulate (asdefined herein) HER-2 or HER-2 mediated signalling by blocking orinhibiting metalloproteinase-mediated HER2 ectodomain shedding (forexample, as determined by the methodology described in Molina et al.2001, Cancer Res. 61: 4744); or more generally capable of modulating (asdefined herein) HER-2 or HER-2 mediated signalling via the samemechanism of action as Herceptin®. Such amino acid sequences andpolypeptides of the invention preferably are directed against theHerceptin® binding site on HER2 and/or capable of competing withHerceptin® for binding to HER-2, and may in particular be directedagainst domain IV of HER2, and more in particular against the C-terminusof domain IV of HER2 (see also Cho et al. (2003), Nature 421:756-760).

In another preferred, but non-limiting aspect, the amino acid sequencesand polypeptides of the invention are directed against the Omnitargbinding site on HER2 and/or are capable of competing with Omnitarg(and/or with the Omnitarg-Fab used in Example 9) for binding to HER-2,as determined using a suitable competition assay, such as the assaydescribed in Example 9. Such amino acid sequences and polypeptides ofthe invention may be as further defined herein. The amino acid sequencesand polypeptides of the invention may be directed against domain II ofHER2. In a preferred aspect, the amino acid sequences and polypeptidesof the invention are directed against the center of domain II of HER2.

In another preferred, but non-limiting aspect, the amino acid sequencesand polypeptides of the invention are capable, upon binding to HER-2, tomodulate (as defined herein) HER-2 or HER-2 mediated signalling byinhibiting ligand activation of an ErbB hetero-oligomer comprising HER2and HER3, HERO or EGFR, or more generally capable of modulating (asdefined herein) HER-2 or HER-2 mediated signalling via the samemechanism of action as Omnitarg. Such amino acid sequences andpolypeptides of the invention preferably are directed against theOmnitarg binding site on HER2 and/or capable of competing with Omnitarg(and/or with the Omnitarg-Fab used in Example 9) for binding to HER-2,and may in particular be directed against domain II of HER2, and more inparticular against the middle of domain II of HER2 (see also Franklin etal. (2004), Cancer cell 5:317-328).

It is also within the scope of the invention that, where applicable, anamino acid sequence or polypeptide of the invention can bind to two ormore antigenic determinants, epitopes, parts, domains, subunits orconfirmations of HER2. In such a case, the antigenic determinants,epitopes, parts, domains or subunits of HER2 to which the amino acidsequences and/or polypeptides of the invention bind may be essentiallythe same (for example, if HER2 contains repeated structural motifs oroccurs in a multimeric form) or may be different (and in the lattercase, the amino acid sequences and polypeptides of the invention maybind to such different antigenic determinants, epitopes, parts, domains,subunits of HER2 with an affinity and/or specificity which may be thesame or different).

In a preferred aspect, the amino acid sequences and (in particular)polypeptides of the invention are capable of binding to two or moredifferent antigenic determinants, epitopes, parts, domains of HER2. Inthis context, the amino acid sequences and polypeptides of the inventionare also referred to as “multiparatopic” (such as e.g. “biparatopic” or“triparatopic”, etc.) amino acid sequences and polypeptides. Themultiparatopic amino acid sequences and polypeptides of the inventioncan be directed against any antigenic determinants, epitopes, parts,and/or domains of HER2.

For example, and generally, a biparatopic polypeptide of the inventionmay comprise at least one amino acid sequence of the invention directedagainst a first antigenic determinant, epitope, part or domain of HER-2and at least one amino acid sequence of the invention directed against asecond antigenic determinant, epitope, part or domain of HER-2 differentfrom the first antigenic determinant, epitope, part or domain (in whichsaid amino acid sequences may be suitably linked, for example via asuitable linker as further described herein). Preferably, such abiparatopic polypeptide of the invention is further such that, when itbinds to HER-2, it is capable of simultaneously binding to the firstantigenic determinant, epitope, part or domain (i.e. via the at leastone amino acid sequence of the invention capable of binding to saidfirst antigenic determinant, epitope, part or domain) and binding tosaid second antigenic determinant, epitope, part or domain (i.e. via theat least one amino acid sequence of the invention capable of binding tosaid second antigenic determinant, epitope, part or domain). Examples ofsuch biparatopic polypeptides of the invention will become clear fromthe further description herein. Also; a triparatopic polypeptide of theinvention may comprise at least one further amino acid sequence of theinvention directed against a third antigenic determinant, epitope, partor domain of HER-2 (different from both the first and second antigenicdeterminant, epitope, part or domain), and generally multiparatopicpolypeptides of the invention may contain at least two amino acidsequences of the invention directed against at least two differentantigenic determinants, epitopes, parts or domains of HER-2. Generally,such biparatopic, triparatopic and multiparatopic polypeptides of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic, triparatopic and multiparatopic polypeptides of theinvention (for example, these biparatopic, triparatopic andmultiparatopic polypeptides of the invention preferably comprise singlevariable domains and more preferably Nanobodies).

In a preferred, but non-limiting aspect, the amino acid sequences and(in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are directed against the Herceptin® binding site onHER2 and/or capable of competing with Herceptin® for binding to HER-2,as well as against at least one other antigenic determinant, epitope,part or domain on HER2. The amino acid sequences and polypeptides of theinvention may be directed against domain IV of HER2 as well as againstat least one other antigenic determinant, epitope, part or domain onHER2. In a preferred aspect, the amino acid sequences and polypeptidesof the invention are directed against the C-terminus of domain IV ofHER2 as well as against at least one other antigenic determinant,epitope, part or domain on. HER2. Generally, such a biparatopic (ormultiparatopic) polypeptide of the invention will contain at least oneamino acid sequence of the invention that is capable of binding to theHerceptin® binding site on HER2 and/or capable of competing withHerceptin® for binding to HER-2 (and in particular against the domain IVof HER-2 and more preferably against the C-terminus of domain IV ofHER2), as well as at least one further amino acid sequence of theinvention that is capable of binding to at least one other antigenicdeterminant, epitope, part or domain on HER2. Generally, suchbiparatopic (or multiparatopic) polypeptides of the invention may be asfurther described herein, and the various preferred aspects of theinvention as described herein also apply to these biparatopic (ormultiparatopic) polypeptides of the invention (for example, thesebiparatopic and multiparatopic polypeptides of the invention maycomprise suitable linkers; are preferably such that they cansimultaneously bind the Herceptin® binding site and the at least oneother antigenic determinant, epitope, part or domain on HER2; andpreferably comprise single variable domains and more preferablyNanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are at least capable, upon binding to HER-2, to (i)recruit immune effector cells such as macrophages and monocytes to thetumor (for this purpose, most preferably a polypeptide of the inventionis used that contains an Fc portion that confers upon the polypeptidethe ability to recruit immune effector cells such as macrophages andmonocytes to the tumor); and/or (ii) modulate (as defined herein) HER-2or HER-2 mediated signalling by downregulating HER2 levels (for example,as determined by the methodology described in Hudziak et al. 1989, Mol.Cell. Biol 9: 1165) and/or by downregulating HER2-mediated signalingpathways (for example, as determined by the methodology described inLane et al. 2000, Mol. Cell. Biol. 20: 3210, Motoyama et al. 2002,Cancer Res. 62: 3151); and/or (iii) modulate (as defined herein) HER-2or HER-2 mediated signalling by blocking or inhibitingmetalloproteinase-mediated HER2 ectodomain shedding (for example, asdetermined by the methodology described in Molina et al. 2001, CancerRes. 61: 4744); or more generally by modulating (as defined herein)HER-2 or HER-2 mediated signalling via the same mechanism of action asHerceptin®.

Such biparatopic (or multiparatopic) polypeptides of the inventionpreferably either (a) comprise an Fc portion that confers upon thepolypeptide the ability to recruit immune effector cells such asmacrophages and monocytes to the tumor), and/or (b) comprise at leastone amino acid sequence of the invention that is capable, upon bindingto HER-2, to (1) modulate (as defined herein) HER-2 or HER-2 mediatedsignalling by downregulating HER2 levels and/or by downregulatingHER2-mediated signaling pathways; and/or (2) modulate (as definedherein) HER-2 or HER-2 mediated signalling by blocking or inhibitingmetalloproteinase-mediated HER2 ectodomain shedding; or more generally(3) modulate (as defined herein) HER-2 or HER-2 mediated signalling viathe same mechanism of action as Herceptin®; as well as at least onefurther amino acid sequence of the invention that is capable of bindingto at least one other antigenic determinant, epitope, part or domain onHER2 (i.e. different from the antigenic determinant, epitope, part ordomain to which the aforementioned amino acid sequence of the inventioncan bind). Such biparatopic (or multiparatopic) polypeptides of theinvention preferably comprise at least one amino acid sequence of theinvention that is directed against the Herceptin® binding site on HER2and/or capable of competing with Herceptin® for binding to HER-2 (and inparticular against domain IV of HER2, and more in particular against theC-terminus of domain IV of HER2), as well as at least one further aminoacid sequence of the invention that is capable of binding to at leastone other antigenic determinant, epitope, part or domain on HER2.Generally, such biparatopic (or multiparatopic) polypeptides of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind the Herceptin® binding site and the at least oneother antigenic determinant, epitope, part or domain on HER2; andpreferably comprise single variable domains and more preferablyNanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand polypeptides of the invention are biparatopic (or multiparatopic)and are directed against the Omnitarg binding site on HER2 and/orcapable of competing with Omnitarg for binding to HER-2, as well asagainst at least one other antigenic determinant on HER2. The amino acidsequences and polypeptides of the invention may be directed againstdomain II of HER2 as well as against at least one other antigenicdeterminant on HER2. In a preferred aspect, the amino acid sequences andpolypeptides of the invention are directed against the center of domainII of HER2 as well as against at least one other antigenic determinanton HER2. Generally, such a biparatopic (or multiparatopic) polypeptideof the invention will contain at least one amino acid sequence of theinvention that is capable of binding to the Omnitarg binding site onHER2 and/or capable of competing with Omnitarg for binding to HER-2 (andin particular against the domain II of HER-2 and more preferably againstthe middle of domain. II of HER2), as well as at least one further aminoacid sequence of the invention that is capable of binding to at leastone other antigenic determinant, epitope, part or domain on HER2.Generally, such biparatopic (or multiparatopic) polypeptides of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind the Omnitarg binding site and the at least oneother antigenic determinant, epitope, part or domain on HER2; andpreferably comprise single variable domains and more preferablyNanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are at least capable, upon binding to HER-2, tomodulate (as defined herein) HER-2 or HER-2 mediated signalling byinhibiting ligand activation of an ErbB hetero-oligomer comprising HER2and HER3, HER4 or EGFR, or more generally capable of modulating (asdefined herein) HER-2 or HER-2 mediated signalling via the samemechanism of action as Omnitarg.

Such biparatopic (or multiparatopic) polypeptides of the inventionpreferably comprise at least one amino acid sequence of the inventionthat is capable, upon binding to HER-2, to modulate (as defined herein)HER-2 or HER-2 mediated signalling by inhibiting ligand activation of anErbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR, or moregenerally capable of modulating (as defined herein) HER-2 or HER-2mediated signalling via the same mechanism of action as Omnitarg; aswell as at least one further amino acid sequence of the invention thatis capable of binding to at least one other antigenic determinant,epitope, part or domain on HER2 (i.e. different from the antigenicdeterminant, epitope, part or domain to which the aforementioned aminoacid sequence of the invention can bind). Such biparatopic (ormultiparatopic) polypeptides of the invention preferably comprise atleast one amino acid sequence of the invention that is directed againstthe Omnitarg binding site on HER2 and/or capable of competing withOmnitarg for binding to HER-2 (and in particular against domain II ofHER2, and more in particular against the middle of domain II of HER2),as well as at least one further amino acid sequence of the inventionthat is capable of binding to at least one other antigenic determinant,epitope, part or domain on HER2. Generally, such biparatopic (ormultiparatopic) polypeptides of the invention may be as furtherdescribed herein, and the various preferred aspects of the invention asdescribed herein also apply to these biparatopic (or multiparatopic)polypeptides of the invention (for example, these biparatopic andmultiparatopic polypeptides of the invention may comprise suitablelinkers; are preferably such that they can simultaneously bind theOmnitarg binding site and the at least one other antigenic determinant,epitope, part or domain on HER2; and preferably comprise single variabledomains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic andare at least directed against the Herceptin® binding site on HER2 aswell as against the Omnitarg binding site on HER2. The amino acidsequences and polypeptides of the invention may be directed againstdomain IV of HER2. The amino acid sequences and polypeptides of theinvention may be directed against domain II of HER2. The amino acidsequences and polypeptides of the invention may be directed againstdomain IV of HER2 as well as against domain II of HER2. In a preferredaspect, the amino acid sequences and polypeptides of the invention aredirected against the C-terminus of domain IV of HER2. In anotherpreferred aspect, the amino acid sequences and polypeptides of theinvention are directed against the C-terminus of domain IV of HER2 aswell as against domain H of HER2. In another preferred aspect, the aminoacid sequences and polypeptides of the invention are directed againstthe center of domain II of HER2. In another preferred aspect, the aminoacid sequences and polypeptides of the invention are directed againstdomain IV of HER2 as well as against the center of domain II of HER2. Inanother preferred aspect, the amino acid sequences and polypeptides ofthe invention are directed against the C-terminus of domain IV of HER2as well as against the center of domain II of HER2.

Again, the above biparatopic (or multiparatopic) polypeptides of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind the Omnitarg binding site and theHerceptin®-binding site; and preferably comprise single variable domainsand more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic withboth paratopes directed against the Herceptin® binding site on HER2. Theamino acid sequences and polypeptides of the invention may be directedagainst domain IV of HER2 (one paratope or both paratopes). In apreferred aspect, the amino acid sequences and polypeptides of theinvention are directed against the C-terminus of domain IV of HER2 (oneparatope or both paratopes).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic withboth paratopes directed against the Omnitarg binding site on HER2. Theamino acid sequences and polypeptides of the invention may be directedagainst domain. II of HER2 (one paratope or both paratopes). In apreferred aspect, the amino acid sequences and polypeptides of theinvention are directed against the center of domain. II of HER2 (oneparatope or both paratopes).

Again, the above biparatopic (or multiparatopic) polypeptides of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind both binding sites; and preferably comprisesingle variable domains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are at least capable, upon binding to HER-2, (A) tomodulate (as defined herein) HER-2 or HER-2 mediated signalling byinhibiting ligand activation of an ErbB hetero-oligomer comprising HER2and HER3, HER4 or EGFR, or more generally capable of modulating (asdefined herein) HER-2 or HER-2 mediated signalling via the samemechanism of action as Omnitarg; and (B) to (i) recruit immune effectorcells such as macrophages and monocytes to the tumor (for this purpose,most preferably a polypeptide of the invention is used that contains anFc portion that confers upon the polypeptide the ability to recruitimmune effector cells such as macrophages and monocytes to the tumor);and/or (ii) modulate (as defined herein) HER-2 or HER-2 mediatedsignalling by downregulating HER2 levels and/or by down-regulatingHER2-mediated signaling pathways; and/or (iii) modulate (as definedherein) HER-2 or HER-2 mediated signalling by blocking or inhibitingmetalloproteinase-mediated HER2 ectodomain shedding; or more generallyby modulating (as defined herein) HER-2 or HER-2 mediated signalling viathe same mechanism of action as Herceptin®.

For example, for this purpose, such a biparatopic (or multiparatopic)polypeptide of the invention may comprise

-   -   at least one first amino acid sequence of the invention that is        capable, upon binding to HER-2, to modulate (as defined herein)        HER-2 or HER-2 mediated signalling by inhibiting ligand        activation of an ErbB hetero-oligomer comprising HER2 and HER3,        HER4 or EGFR, or more generally capable of modulating (as        defined herein) HER-2 or HER-2 mediated signalling via the same        mechanism of action as Omnitarg. Such an amino acid sequence of        the invention is preferably an amino acid sequence that is        directed against the Omnitarg binding site on HER2 (and in        particular against domain II of HER2, and more in particular        against the middle of domain II of HER2) and/or capable of        competing with Omnitarg for binding to HER-2;        and further comprise either    -   an Fc portion that confers upon the polypeptide the ability to        recruit immune effector cells such as macrophages and monocytes        to the tumor),        and/or    -   at least one amino acid sequence of the invention that is        capable, upon binding to HER-2, to (1) modulate (as defined        herein) HER-2 or HER-2 mediated signalling by downregulating        HER2 levels and/or by downregulating HER2-mediated signaling        pathways; and/or (2) modulate (as defined herein) HER-2 or HER-2        mediated signalling by blocking or inhibiting        metalloproteinase-mediated HER2 ectodomain shedding; or more        generally (3) modulate (as defined herein) HER-2 or HER-2        mediated signalling via the same mechanism of action as        Herceptin®. Such an amino acid sequence of the invention is        preferably an amino acid sequence that is directed against the        Herceptin® binding site on HER2 (and in particular against        domain IV of HER2, and more in particular against the C-terminus        of domain IV of HER2) and/or capable of competing with        Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind at least two different antigenic determinants,epitopes, parts or domains or HER-2, such as the Omnitarg binding siteand the Herceptin®-binding site; and preferably comprise single variabledomains and more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are at least capable, upon binding to HER-2, (A) tomodulate (as defined herein) HER-2 or HER-2 mediated signalling byinhibiting ligand activation of an ErbB hetero-oligomer comprising HER2and HER3, HER4 or EGFR, or more generally capable of modulating (asdefined herein) HER-2 or HER-2 mediated signalling via the samemechanism of action as Omnitarg; and (B) to bind to the Herceptin®binding site on HER2 (and in particular to domain IV of HER2, and morein particular to the C-terminus of domain IV of HER2) and/or to competewith Herceptin® for binding to HER-2.

For example, for this purpose, such a biparatopic (or multiparatopic)polypeptide of the invention may comprise

-   -   at least one first amino acid sequence of the invention that is        capable, upon binding to HER-2, to modulate (as defined herein)        HER-2 or HER-2 mediated signalling by inhibiting ligand        activation of an ErbB hetero-oligomer comprising HER2 and HER3,        HER4 or EGFR, or more generally capable of modulating (as        defined herein) HER-2 or HER-2 mediated signalling via the same        mechanism of action as Omnitarg;        and further comprise either    -   at least one amino acid sequence of the invention that is        directed against the Herceptin® binding site on HER2 (and in        particular against domain IV of HER2, and more in particular        against the C-terminus of domain IV of HER2) and/or capable of        competing with Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind at least two different antigenic determinants,epitopes, parts or domains or HER-2, at least including theHerceptin®-binding site; and preferably comprise single variable domainsand more preferably Nanobodies).

In another preferred, but non-limiting aspect, the amino acid sequencesand (in particular) polypeptides of the invention are biparatopic (ormultiparatopic) and are at least capable, upon binding to HER-2, (A) tobind to the Omnitarg binding site on HER2 (and in particular to domainII of HER2, and more in particular to the middle of domain II of HER2)and/or capable of competing with Omnitarg for binding to HER-2; and (B)to (i) recruit immune effector cells such as macrophages and monocytesto the tumor (for this purpose, most preferably a polypeptide of theinvention is used that contains an Fc portion that confers upon thepolypeptide the ability to recruit immune effector cells such asmacrophages and monocytes to the tumor); and/or (ii) modulate (asdefined herein) HER-2 or HER-2 mediated signalling by downregulatingHER2 levels and/or by downregulating HER2-mediated signaling pathways;and/or (iii) modulate (as defined herein) HER-2 or HER-2 mediatedsignalling by blocking or inhibiting metalloproteinase-mediated HER2ectodomain shedding; or more generally by modulating (as defined herein)HER-2 or HER-2 mediated signalling via the same mechanism of action asHerceptin®.

For example, for this purpose, such a biparatopic (or multiparatopic)polypeptide of the invention may comprise:

-   -   at least one first amino acid sequence of the invention that is        directed against the Omnitarg binding site on HER2 (and in        particular against domain II of HER2, and more in particular        against the middle of domain II of HER2) and/or capable of        competing with Omnitarg for binding to HER-2;        and further comprise either    -   an Fc portion that confers upon the polypeptide the ability to        recruit immune effector cells such as macrophages and monocytes        to the tumor),        and/or    -   at least one amino acid sequence of the invention that is        capable, upon binding to HER-2, to (1) modulate (as defined        herein) HER-2 or HER-2 mediated signalling by downregulating        HER2 levels and/or by downregulating HER2-mediated signaling        pathways; and/or (2) modulate (as defined herein) HER-2 or HER-2        mediated signalling by blocking or inhibiting        metalloproteinase-mediated HER2 ectodomain shedding; or more        generally (3) modulate (as defined herein) HER-2 or HER-2        mediated signalling via the same mechanism of action as        Herceptin®. Such an amino acid sequence of the invention is        preferably an amino acid sequence that is directed against the        Herceptin® binding site on HER2 (and in particular against        domain IV of HER2, and more in particular against the C-terminus        of domain IV of HER2) and/or capable of competing with        Herceptin® for binding to HER-2.

Again, such a biparatopic (or multiparatopic) polypeptide of theinvention may be as further described herein, and the various preferredaspects of the invention as described herein also apply to thesebiparatopic (or multiparatopic) polypeptides of the invention (forexample, these biparatopic and multiparatopic polypeptides of theinvention may comprise suitable linkers; are preferably such that theycan simultaneously bind at least two different antigenic determinants,epitopes, parts or domains or HER-2, at least including the Omnitargbinding site; and preferably comprise single variable domains and morepreferably Nanobodies).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofHER2; or at least to those analogs, variants, mutants, alleles, partsand fragments of HER2 that contain one or more antigenic determinants orepitopes that are essentially the same as the antigenic determinant(s)or epitope(s) to which the amino acid sequences and polypeptides of theinvention bind in HER2 (e.g. in wild-type HER2). Again, in such a case,the amino acid sequences and polypeptides of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e. higher than or lower than), the affinity and specificity withwhich the amino acid sequences of the invention bind to (wild-type)HER2. It is also included within the scope of the invention that theamino acid sequences and polypeptides of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of HER2, butnot to others.

When HER2 exists in a monomeric form and in one or more multimericforms, it is within the scope of the invention that the amino acidsequences and polypeptides of the invention only bind to HER2 inmonomeric form, only bind to HER2 in multimeric form, or bind to boththe monomeric and the multimeric form. Again, in such a case, the aminoacid sequences and polypeptides of the invention may bind to themonomeric form with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to the multimeric form.

In a non-limiting aspect, the amino acid sequences and polypeptides ofthe invention only bind to HER2 in monomeric form while not binding toHER2 in dimerized state. In another non-limiting aspect, the amino acidsequences and polypeptides of the invention only bind to HER2 indimerized state while not binding to HER2 in monomeric form. In anothernon-limiting aspect, the amino acid sequences and polypeptides of theinvention bind to HER2 in monomeric form as well as to HER2 in dimerizedstate.

Also, when HER2 can associate with other proteins or polypeptides (e.g.with other ERBB receptors, also referred to as heterodimerization) toform protein complexes (e.g. with multiple subunits), it is within thescope of the invention that the amino acid sequences and polypeptides ofthe invention bind to HER2 in its non-associated state, bind HER2 in itsassociated state, or bind to both. In a non-limiting aspect, the aminoacid sequences and polypeptides of the invention only bind to HER2 whenHER-2 is in its monomeric form while not binding to HER2 when HER-2 isin its dimerized state. In another non-limiting aspect, the amino acidsequences and polypeptides of the invention only bind to HER2 when HER-2is in its dimerized state while not binding to HER2 when HER-2 is inmonomeric form. In another non-limiting aspect, the amino acid sequencesand polypeptides of the invention bind to HER2 in monomeric form as wellas to HER2 in dimerized state. In all these cases, the amino acidsequences and polypeptides of the invention may bind to such multimersor associated protein complexes with an affinity and/or specificity thatmay be the same as or different from (i.e. higher than or lower than)the affinity and/or specificity with which the amino acid sequences andpolypeptides of the invention bind to HER2 in its monomeric andnon-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed against HER2 maybind with higher avidity to HER2 than the corresponding monomeric aminoacid sequence(s). For example, and without limitation, proteins orpolypeptides that contain two or more amino acid sequences directedagainst different epitopes of HER2 may (and usually will) bind withhigher avidity than each of the different monomers, and proteins orpolypeptides that contain two or more amino acid sequences directedagainst HER2 may (and usually will) bind also with higher avidity to amultimer of HER2.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of HER2 (including monomeric, multimericand associated forms) that are the most relevant from a biologicaland/or therapeutic point of view, as will be clear to the skilledperson.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against HER2; and more preferably willbe capable of specific binding to HER2, and even more preferably capableof binding to HER2 with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein) that is as defined herein. Somenon-limiting examples of such parts, fragments, analogs, mutants,variants, alleles, derivatives, proteins and/or polypeptides will becomeclear from the further description herein. Additional fragments orpolypeptides of the invention may also be provided by suitably combining(i.e. by linking or genetic fusion) one or more (smaller) parts orfragments as described herein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to HER2; and more preferably capable of binding to HER2 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such amino acidsequences are preferably such that they comprise an immunoglobulin foldor are capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called. V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody® (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody® (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N.V.] Such Nanobodies directed against HER2 will also be referredto herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against HER2, and for example alsocovers the Nanobodies belonging to the so-called “V_(H)4 class” (i.e.Nanobodies with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin WO 07/118,670.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below;    and in which:

-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NOs: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NOs: 1 to 22) are    disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against HER2, to suitable fragmentsthereof, as well as to polypeptides that comprise or essentially consistof one or more of such Nanobodies and/or suitable fragments.

SEQ ID NOs: 2051-2325 give the amino acid sequences of a number ofV_(HH) sequences that have been raised against HER2.

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined        herein) HER2 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 2051-2325. These        amino acid sequences may further be such that they are directed        against an interaction site (as defined herein) on HER2 (such as        the Herceptin® or Omnitarg binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 2051-2325 to HER2 and/or that compete with at least one of        the amino acid sequences of SEQ ID NO's: 2051-2325 for binding        to HER2. Again, these amino acid sequences may further be such        that they are directed against an interaction site (as defined        herein) on HER2 (such as Herceptin® or Omnitarg binding site);

which amino acid sequences may be as further described herein (and mayfor example be Nanobodies); as well as polypeptides of the inventionthat comprise one or more of such amino acid sequences (which may be asfurther described herein, and may for example be bispecific and/orbiparatopic polypeptides as described herein), and nucleic acidsequences that encode such amino acid sequences and polypeptides. Suchamino acid sequences and polypeptides do not include any naturallyoccurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to HER2 and which:

-   i) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NOs: 2051-2325, in which for the    purposes of determining the degree of amino acid identity, the amino    acid residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-1, which lists the    framework 1 sequences (SEQ ID NOs: 126-400), framework 2 sequences    (SEQ ID NOs: 676-950), framework 3 sequences (SEQ ID NOs: 1226-1500)    and framework 4 sequences (SEQ ID NOs: 1776-2050) of the Nanobodies    of SEQ ID NOs: 2051-2325 (with respect to the amino acid residues at    positions 1 to 4 and 27 to 30 of the framework 1 sequences,    reference is also made to the comments made below. Thus, for    determining the degree of amino acid identity, these residues are    preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said sequence (in any manner known per se, as furtherdescribed herein) and the resulting humanized V_(HH) sequences can betested for affinity for the target, for stability, for ease and level ofexpression, and/or for other desired properties. In this way, by meansof a limited degree of trial and error, other suitable humanizingsubstitutions (or suitable combinations thereof) can be determined bythe skilled person based on the disclosure herein. Also, based on theforegoing, (the framework regions of) a Nanobody may be partiallyhumanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention arehumanized variants of the Nanobodies of SEQ ID NOs: 2051-2325.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to HER2 and which:

-   i) are a humanized variant of one of the amino acid sequences of SEQ    ID NOs: 2051-2325; and/or-   ii) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NOs: 2051-2325, in which for the    purposes of determining the degree of amino acid identity, the amino    acid residues that form the CDR sequences are disregarded;    and in which:-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to HER2. Thesestretches of amino acid residues may be present in, and/or may becorporated into, an amino acid sequence of the invention, in particularin such a way that they form (part of) the antigen binding site of anamino acid sequence of the invention. As these stretches of amino acidresidues were first generated as CDR sequences of heavy chain antibodiesor V_(HH) sequences that were raised against HER2 (or may be based onand/or derived from such CDR sequences, as further described herein),they will also generally be referred to herein as “CDR sequences” (i.e.as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). Itshould however be noted that the invention in its broadest sense is notlimited to a specific structural role or function that these stretchesof amino acid residues may have in an amino acid sequence of theinvention, as long as these stretches of amino acid residues allow theamino acid sequence of the invention to bind to HER2. Thus, generally,the invention in its broadest sense comprises any amino acid sequencethat is capable of binding to HER2 and that comprises one or more CDRsequences as described herein, and in particular a suitable combinationof two or more such CDR sequences, that are suitably linked to eachother via one or more further amino acid sequences, such that the entireamino acid sequence forms a binding domain and/or binding unit that iscapable of binding to HER2. It should however also be noted that thepresence of only one such CDR sequence in an amino acid sequence of theinvention may by itself already be sufficient to provide an amino acidsequence of the invention that is capable of binding to HER2; referenceis for example again made to the so-called “Expedite fragments”described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to HER2, and more in particular such that itcan bind to HER2 with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against HER2, that comprises one or morestretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 401-675;-   ii) the amino acid sequences of SEQ ID NO's: 951-1225; and-   iii) the amino acid sequences of SEQ ID NO's: 1501-1775;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against HER2.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against HER2, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), g), h) or i); (ii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to d), e) or f), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), g), h) or i); or (iii) when the    first stretch of amino acid residues corresponds to one of the amino    acid sequences according to g), h) or i), the second stretch of    amino acid residues corresponds to one of the amino acid sequences    according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 401-675;-   ii) the amino acid sequences of SEQ ID NO's: 951-1225; and-   iii) the amino acid sequences of SEQ ID NO's: 1501-1775;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    401-675, the second stretch of amino acid residues corresponds to    one of the amino acid sequences of SEQ ID NO's: 951-1225 or of SEQ    ID NO's: 1501-1775; (ii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 951-1225, the second stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    401-675 or of SEQ ID NO's: 1501-1775; or (iii) when the first    stretch of amino acid residues corresponds to one of the amino acid    sequences of SEQ ID NO's: 1501-1775, the second stretch of amino    acid residues corresponds to one of the amino acid sequences of SEQ    ID NO's: 401-675 or of SEQ ID NO's: 951-1225.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against HER2.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against HER2, that comprises three ormore stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with, at least one of the amino acid sequences of SEQ ID NO's:    401-675;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 401-675; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 951-1225; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:1501-1775.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against HER2. Preferred combinations of such stretches ofamino acid sequences will become clear from the further disclosureherein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 2051-2325. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said amino acid sequence and one or more ofthe sequences of SEQ ID NO's: 2051-2325, in which the amino acidresidues that form the framework regions are disregarded. Also, suchamino acid sequences of the invention can be as further describedherein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to HER2; and more in particularbind to HER2 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 401-675; and/or CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 951-1225; and/orCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 1501-1775.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775; or any suitable fragment of such an amino acid sequence.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 401-675 and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 951-1225; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 1501-1775.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to HER2; and more in particularbind to HER2 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 2051-2325. This degree of aminoacid identity can for example be determined by determining the degree ofamino acid identity (in a manner described herein) between said aminoacid sequence and one or more of the sequences of SEQ ID NO's:2051-2325, in which the amino acid residues that form the frameworkregions are disregarded. Such amino acid sequences of the invention canbe as further described herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody® (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody®. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531, aswell as to WO 08/068,280 of Ablynx N.V.

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin; see forexample EP 0 368 684 B1, page 4); or polypeptides of the invention thatcomprise at least one amino acid sequence of the invention that islinked to at least one moiety (and in particular at least one amino acidsequence) that increases the half-life of the amino acid sequence of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or amino acid sequences will become clearto the skilled person based on the further disclosure herein; and forexample include, without limitation, polypeptides in which the one ormore amino acid sequences of the invention are suitable linked to one ormore serum proteins or fragments thereof (such as (human) serum albuminor suitable fragments thereof) or to one or more binding units that canbind to serum proteins (such as, for example, domain antibodies, aminoacid sequences that are suitable for use as a domain antibody, singledomain antibodies, amino acid sequences that are suitable for use as asingle domain antibody, “dAb”'s, amino acid sequences that are suitablefor use as a dAb, or Nanobodies that can bind to serum proteins such asserum albumin (such as human serum albumin), serum immunoglobulins suchas IgG, or transferrine; reference is made to the further descriptionand references mentioned herein); polypeptides in which an amino acidsequence of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more amino acid sequences of the invention are suitable linked toone or more small, proteins or peptides that can bind to serum proteins(such as, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280 of Ablynx N.V.

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereat).

Such a nucleic acid will also be referred to herein as a “nucleic acidof the invention” and may for example be in the form of a geneticconstruct, as further described herein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating HER2, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or in a multicellular organism, and in particular in amammal, and more in particular in a human being, such as in a humanbeing that is at risk of or suffers from a cancer and/or tumor); and/orin methods for killing a tumor cell or inhibiting or preventingproliferation of a tumour cell (either in vitro or in vivo) by suitablycontacting said tumor cell with an amino acid sequence, Nanobody orpolypeptide of the invention, or of a composition comprising the same.In a preferred, but non-limiting aspect, a biparatopic (ormultiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

The invention also relates to methods for modulating HER2, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a cancer and/or tumor), which methodcomprises at least the step of contacting HER2 with at least one aminoacid sequence, Nanobody or polypeptide of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate HER2, with at least one amino acid sequence, Nanobody orpolypeptide of the invention. In a preferred, but non-limiting aspect, abiparatopic (or multiparatopic) polypeptide of the invention is used,and more preferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating HER2, eitherin vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in ana single cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a cancer and/or tumor). In a preferred,but non-limiting aspect, a biparatopic (or multiparatopic) polypeptideof the invention is used, and more preferably one of the preferredbiparatopic (or multiparatopic) polypeptides of the invention, asfurther described herein.

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, HER2, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein). In particular, “modulating” or “to modulate” may mean eitherreducing or inhibiting the activity of, or alternatively increasing theactivity of HER2, as measured using a suitable in vitro, cellular or invivo assay (such as those mentioned herein), by at least 1%, preferablyat least 5%, such as at least 10% or at least 25%, for example by atleast 50%, at least 60%, at least 70%, at least 80%, or 90% or more,compared to activity of HER2 in the same assay under the same conditionsbut without the presence of the amino acid sequence, Nanobody orpolypeptide of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inthe sensitivity of HER2 for one or more conditions in the medium orsurroundings in which HER2 is present (such as pH, ion strength, thepresence of co-factors, etc.), compared to the same conditions butwithout the presence of the amino acid sequence, Nanobody or polypeptideof the invention. As will be clear to the skilled person, this may againbe determined in any suitable manner and/or using any suitable assayknown per se, such as the assays described herein or in the prior artcited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which HER2 (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, such as the assays described herein orin the prior art cited herein. In particular, an action as an agonist orantagonist may be such that an intended biological or physiologicalactivity is increased or decreased, respectively, by at least 1%,preferably at least 5%, such as at least 10% or at least 25%, forexample by at least 50%, at least 60%, at least 70%, at least 80%, or90% or more, compared to the biological or physiological activity in thesame assay under the same conditions but without the presence of theamino acid sequence, Nanobody or polypeptide of the invention.

Modulating may also involve activating HER2 or the mechanism or pathwayin which it is involved. Modulating may be reversible or irreversible,but for pharmaceutical and pharmacological purposes will usually be in areversible manner. Modulating may for example also involve reducing orinhibiting the binding of HER2 to another ERBB receptor (also referredto as heterodimerization) and/or competing with another ERBB receptorfor binding to HER2.

Without being limiting, in one aspect, the amino acid sequence, Nanobodyor polypeptide of the invention or the composition comprising the samewill inhibit and/or block binding of Herceptin® to HER2. The amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will preferably inhibit binding of Herceptin® toHER2 by at least 1%, preferably at least 5%, such as at least 10%, forexample 25% or more or even 50% or more and up to 75% or even more than90% or more, compared to binding of Herceptin® to HER2 in the absence ofthe amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same.

In another aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock binding of Omnitarg to HER2. The amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willpreferably inhibit binding of Omnitarg to HER2 by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto binding of Omnitarg to HER2 in the absence of the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same.

In another aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock binding of Herceptin® and Omnitarg to HER, preferably essentiallysimultaneously. The amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will preferably inhibitbinding of Herceptin® to HER2 by at least 1%, preferably at least 5%,such as at least 10%, for example 25% or more or even 50% or more and upto 75% or even more than 90% or more, compared to binding of Herceptin®to HER2 in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same; andthe amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will preferably inhibit binding ofOmnitarg to HER2 by at least 1%, preferably at least 5%, such as atleast 10%, for example 25% or more or even 50% or more and up to 75% oreven more than 90% or more, compared to binding of Omnitarg to HER2 inthe absence of the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and/or of the signalling that ismediated by HER-2 and/or by the ligand(s) of HER-2 (i.e. of thesignalling that is caused by binding of growth factors of the EGF familyto HER-2) and will inhibit and/or block such signalling (i.e. by atleast 1%, preferably at least 5%, such as at least 10%, for example 25%or more or even 50% or more and up to 75% or even more than 90% or more,compared to the signalling without the presence of the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same, as determined in a suitable assay); and/or willinhibit or block tumor (e.g. SKBR3) cell proliferation. The amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will preferably inhibit tumor (e.g. SKBR3) cellproliferation by at least 1%, preferably at least 5%, such as at least10%, for example 25% or more or even 50% or more and up to 75% or evenmore than 90% or more, compared to the tumor (e.g. SKBR3) cellproliferation in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same. Ina preferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock tumor (e.g. SKBR3) cell proliferation equally or better thanHerceptin®. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block tumor (e.g. SKBR3) cell proliferationequally or better than Omnitarg. In another preferred aspect, the aminoacid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block tumor (e.g.SKBR3) cell proliferation equally or better than Herceptin® andOmnitarg. In a preferred, but non-limiting aspect, a suitableantagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of diseases and disorders that can be preventedor treated by increasing HER-2 signalling in one or more cells ortissues of a patient to be treated, such as certain cardiac disorders(i.e. those characterised by reduced HER-2-mediated signalling or thosethat are a side-effect from treating a patient with a HER-2 antagonist),the amino acid sequence. Nanobody or polypeptide of the invention or thecomposition comprising the same is an agonist of HER2 and will inducecell proliferation. The amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will preferablyincrease the signalling that is mediated by HER-2 and/or by theligand(s) of HER-2 (i.e. by at least 1%, preferably at least 5%, such asat least 10%, for example 25% or more or even 50% or more and up to 75%or even more than 90% or more, compared to the signalling without thepresence of the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same, as determined in asuitable assay); and/or will induce cell proliferation by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto the cell proliferation in the absence of the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same. In a preferred, but non-limiting aspect, a suitable agonisticbiparatopic (or multiparatopic) polypeptide of the invention is used,and more preferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit, downregulate and/orblock cell signalling. The amino acid sequence, Nanobody or polypeptideof the invention or the composition comprising the same will preferablyinhibit and/or downregulate cell signalling by at least 1%, preferablyat least 5%, such as at least 10%, for example 25% or more or even 50%or more and up to 75% or even more than 90% or more, compared to thecell signalling in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same. Ina preferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit,downregulate and/or block cell signalling equally or better thanHerceptin®. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit, downregulate and/or block cell signalling equallyor better than Omnitarg. In another preferred aspect, the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will inhibit, downregulate and/or block cellsignalling equally or better than Herceptin® and Omnitarg. In apreferred, but non-limiting aspect, a suitable antagonistic biparatopic(or multiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of diseases and disorders that can be preventedor treated by increasing HER-2 signalling in one or more cells ortissues of a patient to be treated, such as certain cardiac disorders(i.e. those characterised by reduced HER-2-mediated signalling or thosethat are a side-effect from treating a patient with a HER-2 antagonist),the amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same is an agonist of HER2 and will inducecell signalling. The amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will preferably inducecell signalling by at least 1%, preferably at least 5%, such as at least10%, for example 25% or more or even 50% or more and up to 75% or evenmore than 90% or more, compared to the cell signalling in the absence ofthe amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same. In a preferred, but non-limitingaspect, a suitable agonistic biparatopic (or multiparatopic) polypeptideof the invention is used, and more preferably one of the preferredbiparatopic (or multiparatopic) polypeptides of the invention, asfurther described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit and/or block tumor(e.g. SKBR3) cell proliferation in vivo. The amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will preferably inhibit tumor (e.g. SKBR3) cell proliferationin vivo by at least 1%, preferably at least 5%, such as at least 10%,for example 25% or more or even 50% or more and up to 75% or even morethan 90% or more, compared to the tumor (e.g. SKBR3) cell proliferationin vivo in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same. Ina preferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock tumor (e.g. SKBR3) cell proliferation in vivo equally or betterthan Herceptin®. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block tumor (e.g. SKBR3) cell proliferationin vivo equally or better than Omnitarg. In another preferred aspect,the amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block tumor (e.g.SKBR3) cell proliferation in vivo equally or better than Herceptin® andOmnitarg. In a preferred, but non-limiting aspect, a suitableantagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit, downregulate and/orblock ligand-mediated ErbB signalling. The amino acid sequence, Nanobodyor polypeptide of the invention or the composition comprising the samewill preferably inhibit and/or downregulate ligand-mediated ErbBsignalling by at least 1%, preferably at least 5%, such as at least 10%,for example 25% or more or even 50% or more and up to 75% or even morethan 90% or more, compared to the ligand-mediated ErbB signalling in theabsence of the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same. In a preferred aspect,the amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or blockligand-mediated ErbB signalling equally or better than Herceptin®. Inanother preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinhibit and/or block ligand-mediated ErbB signalling equally or betterthan Omnitarg. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block ligand-mediated ErbB signallingequally or better than Herceptin® and Omnitarg. In a preferred, butnon-limiting aspect, a suitable antagonistic biparatopic (ormultiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit and/or block HER2ectodomain cleavage. The amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will preferablyinhibit HER2 ectodomain cleavage by at least 1%, preferably at least 5%,such as at least 10%, for example 25% or more or even 50% or more and upto 75% or even more than 90% or more, compared to the HER2 ectodomaincleavage in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same. Ina preferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock HER2 ectodomain cleavage equally or better than Herceptin®. Inanother preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinhibit and/or block HER2 ectodomain cleavage equally or better thanOmnitarg. In another preferred aspect, the amino acid sequence, Nanobodyor polypeptide of the invention or the composition comprising the samewill inhibit and/or block HER2 ectodomain cleavage equally or betterthan Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, asuitable antagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit and/or blockHeregulin-mediated activation of MAPK/Erk1/2. The amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will preferably inhibit Heregulin-mediated activation ofMAPK/Erk1/2 by at least 1%, preferably at least 5%, such as at least10%, for example 25% or more or even 50% or more and up to 75% or evenmore than 90% or more, compared to the Heregulin-mediated activation ofMAPK/Erk1/2 in the absence of the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same. Ina preferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock Heregulin-mediated activation of MAPK/Erk1/2 equally or betterthan Herceptin®. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block Heregulin-mediated activation ofMAPK/Erk1/2 equally or better than Omnitarg. In another preferredaspect, the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will inhibit and/orblock Heregulin-mediated activation of MAPK/Erk1/2 equally or betterthan Herceptin® and Omnitarg. In a preferred, but non-limiting aspect, asuitable antagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit and/or block PI3K/Aktsignalling. The amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will preferably inhibitPI3K/Akt signalling by at least 1%, preferably at least 5%, such as atleast 10%, for example 25% or more or even 50% or more and up to 75% oreven more than 90% or more, compared to the PI3K/Akt signalling in theabsence of the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same. In a preferred aspect,the amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block PI3K/Aktsignalling equally or better than Herceptin®. In another preferredaspect, the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will inhibit and/orblock. PI3K/Akt signalling equally or better than Omnitarg. In anotherpreferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock PI3K/Akt signalling equally or better than Herceptin® andOmnitarg. In a preferred, but non-limiting aspect, a suitableantagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same is an antagonist of HER2 and will inhibit, downregulate and/orblock cell signalling in vivo. The amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willpreferably inhibit and/or downregulate cell signalling in vivo by atleast 1%, preferably at least 5%, such as at least 10%, for example 25%or more or even 50% or more and up to 75% or even more than 90% or more,compared to the cell signalling in vivo in the absence of the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same. In a preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block cell signalling in vivo equally orbetter than Herceptin®. In another preferred aspect, the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will inhibit and/or block cell signalling in vivoequally or better than Omnitarg. In another preferred aspect, the aminoacid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block cellsignalling in vivo equally or better than Herceptin® and Omnitarg. In apreferred, but non-limiting aspect, a suitable antagonistic biparatopic(or multiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will induce apoptosis in tumor cells. The amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will preferably induce apoptosis in tumor cells by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto apoptosis in tumor cells in the absence of the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same. In a preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinduce apoptosis in tumor cells equally or better than Herceptin®. Inanother preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinduce apoptosis in tumor cells equally or better than Omnitarg. Inanother preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinduce apoptosis in tumor cells equally or better than Herceptin® andOmnitarg. In a preferred, but non-limiting aspect, a suitableantagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block heterodimerization between ERBBreceptors. The amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will preferably inhibitand/or block heterodimerization between ERBB receptors by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto the heterodimerization between ERBB receptors in the absence of theamino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same. In a preferred aspect, the amino acidsequence. Nanobody or polypeptide of the invention or the compositioncomprising the same will inhibit and/or block heterodimerization betweenERBB receptors equally or better than Omnitarg. In a preferred, butnon-limiting aspect, a suitable antagonistic biparatopic (ormultiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block tumor vascularisation. The amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will preferably inhibit tumor vascularisation by atleast 1%, preferably at least 5%, such as at least 10%, for example 25%or more or even 50% or more and up to 75% or even more than 90% or more,compared to the tumor vascularisation in the absence of the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same. In a preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block tumor vascularisation equally orbetter than Herceptin®. In another preferred aspect, the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same will inhibit and/or block tumor vascularisationequally or better than Omnitarg. In another preferred aspect, the aminoacid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block tumorvascularisation equally or better than Herceptin® and Omnitarg. In apreferred, but non-limiting aspect, a suitable antagonistic biparatopic(or multiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block TNF induced signalling and/or cellproliferation. The amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will preferably inhibitTNF induced signalling and/or cell proliferation by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto the TNF induced signalling and/or cell proliferation in the absenceof the amino acid sequence, Nanobody or polypeptide of the invention orthe composition comprising the same. In a preferred aspect, the aminoacid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block TNF inducedsignalling and/or cell proliferation equally or better than Herceptin®.In another preferred aspect, the amino acid sequence, Nanobody orpolypeptide of the invention or the composition comprising the same willinhibit and/or block TNF induced signalling and/or cell proliferationequally or better than Omnitarg. In another preferred aspect, the aminoacid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or block TNF inducedsignalling and/or cell proliferation equally or better than Herceptin®and Omnitarg. In a preferred, but non-limiting aspect, a suitableantagonistic biparatopic (or multiparatopic) polypeptide of theinvention is used, and more preferably one of the preferred biparatopic(or multiparatopic) polypeptides of the invention, as further describedherein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will downregulate HER2 levels. The amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will preferably will downregulate HER2 levels by at least 1%,preferably at least 5%, such as at least 10%, for example 25% or more oreven 50% or more and up to 75% or even more than 90% or more, comparedto the HER2 levels in the absence of the amino acid sequence, Nanobodyor polypeptide of the invention or the composition comprising the same.In a preferred aspect, the amino acid sequence, Nanobody or polypeptideof the invention or the composition comprising the same willdown-regulate HER2 levels equally or better than Herceptin®. In anotherpreferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will downregulateHER2 levels equally or better than Omnitarg. In another preferredaspect, the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same will downregulate HER2levels equally or better than Herceptin® and Omnitarg. In a preferred,but non-limiting aspect, a suitable antagonistic biparatopic (ormultiparatopic) polypeptide of the invention is used, and morepreferably one of the preferred biparatopic (or multiparatopic)polypeptides of the invention, as further described herein.

In another aspect, which is for example preferred for use in theprevention and treatment of tumors and cancer, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block metalloproteinase-mediated HER2ectodomain shedding. The amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will preferablyinhibit metalloproteinase-mediated HER2 ectodomain shedding by at least1%, preferably at least 5%, such as at least 10%, for example 25% ormore or even 50% or more and up to 75% or even more than 90% or more,compared to the metalloproteinase-mediated HER2 ectodomain shedding inthe absence of the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same. In a preferred aspect,the amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same will inhibit and/or blockmetalloproteinase-mediated HER2 ectodomain shedding equally or betterthan Herceptin®. In another preferred aspect, the amino acid sequence,Nanobody or polypeptide of the invention or the composition comprisingthe same will inhibit and/or block metalloproteinase-mediated HER2ectodomain shedding equally or better than Omnitarg. In anotherpreferred aspect, the amino acid sequence, Nanobody or polypeptide ofthe invention or the composition comprising the same will inhibit and/orblock metalloproteinase-mediated HER2 ectodomain shedding equally orbetter than Herceptin® and Omnitarg. In a preferred, but non-limitingaspect, a suitable antagonistic biparatopic (or multiparatopic)polypeptide of the invention is used, and more preferably one of thepreferred biparatopic (or multiparatopic) polypeptides of the invention,as further described herein.

The amino acid sequence, Nanobody or polypeptide of the invention or thecomposition comprising the same should at least “modulate” or effect achange (i.e. an activity as an agonist or as an antagonist,respectively) with respect to at least one biological or physiologicalmechanisms, effects, responses, functions, pathways or activities (alsoreferred to herein as “having at least one mode of action”) in whichHER2 (or in which its pathway(s) are involved, such as its signallingpathway or metabolic pathway and their associated biological orphysiological effects) is involved. In one aspect, the amino acidsequence, Nanobody or polypeptide of the invention or the compositioncomprising the same may “modulate” or effect a change with respect tomore than one (such as two, three, four or even more) biological orphysiological mechanisms, effects, responses, functions, pathways oractivities (i.e. the amino acid sequence, Nanobody or polypeptide of theinvention or the composition comprising the same may have more than onemode of action). In this respect, the present inventors surprisinglyfound that the biparatopic amino acid sequence, Nanobody or polypeptideof the invention or the composition comprising the same could combinetwo different modes of action (such as e.g. they could inhibit and/orblock two different cell signalling pathways; they could e.g. inhibitand/or block heterodimerization between ERBB receptors and at the sametime downregulate HER2 levels).

The different modes of action are mediated each by one of the bindingunits (as further defined herein) of the biparatopic amino acidsequence, Nanobody or polypeptide of the invention, wherein each bindingunit binds at a different binding site of HER2. In a preferred aspect,the biparatopic amino acid sequence, Nanobody or polypeptide of theinvention combine the modes of action of Herceptin® and Omnitarg.

Accordingly, the present invention also relates to a biparatopic aminoacid sequence, Nanobody or polypeptide of the invention or a compositioncomprising the same that combines two different modes of action eachmediated by one of the binding units of the biparatopic amino acidsequence, Nanobody or polypeptide of the invention, wherein each bindingunit binds at a different binding site of HER2.

Accordingly, the present invention also relates to a triparatopic aminoacid sequence, Nanobody or polypeptide of the invention or a compositioncomprising the same that combines two or three different modes of actioneach mediated by one of the binding units of the triparatopic amino acidsequence, Nanobody or polypeptide of the invention, wherein each bindingunit binds at a different binding site of HER2.

More generally, the present invention relates to a multiparatopic aminoacid sequence, Nanobody or polypeptide of the invention or a compositioncomprising the same that combines two or more different modes of actioneach mediated by one of the binding units of the multiparatopic aminoacid sequence, Nanobody or polypeptide of the invention, wherein eachbinding unit binds at a different binding site of HER2.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    HER2;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for HER2.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with HER2 or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In the above step b), the set, collection or library may for example bescreened for (nucleic acid sequences that encode) amino acid sequencesthat can bind to the Herceptin® binding site on HER-2 (and may inparticular to domain IV of HER2, more in particular to the C-terminus ofdomain IV of HER2) and/or that compete with Herceptin® for binding toHER-2.

Alternatively, in the above step b), the set, collection or library mayfor example be screened for (nucleic acid sequences that encode) aminoacid sequences that can bind to the Omnitarg binding site on HER-2 (andmay in particular to domain II of HER2, more in particular to the middleof domain II of HER2) and/or that compete with Omnitarg for binding toHER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for HER2;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withHER2 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

Again, in the above step b), the set, collection or library may forexample be screened for (nucleic acid sequences that encode) amino acidsequences that can bind to the Herceptin® binding site on HER-2 (and mayin particular to domain IV of HER2, more in particular to the C-terminusof domain IV of HER2) and/or that compete with Herceptin® for binding toHER-2; or alternatively for (nucleic acid sequences that encode) aminoacid sequences that can bind to the Omnitarg binding site on HER-2 (andmay in particular to domain II of HER2, more in particular to the middleof domain II of HER2) and/or that compete with Omnitarg for binding toHER-2.

In another aspect, the method for generating an amino acid sequencedirected against HER2 may comprise at least the steps of

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for HER2;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

Again, in the above step b), the set, collection or library may forexample be screened for (nucleic acid sequences that encode) amino acidsequences that can bind to the Herceptin® binding site on HER-2 (and mayin particular to domain IV of HER2, more in particular to the C-terminusof domain IV of HER2) and/or that compete with Herceptin® for binding toHER-2; or alternatively for (nucleic acid sequences that encode) aminoacid sequences that can bind to the Omnitarg binding site on HER-2 (andmay in particular to domain II of HER2, more in particular to the middleof domain II of HER2) and/or that compete with Omnitarg for binding toHER-2.

In another aspect, the method for generating an amino acid sequencedirected against HER2 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for HER2 and that is    cross-blocked or is cross blocking a Nanobody of the invention, e.g.    SEQ ID NO: 2051-2325, or a polypeptide or construct of the    invention, e.g. SEQ ID NO: 2326-2390; and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 bindingamino acid sequence fused to the set, collection or library ofnucleotide sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in. NatureBiotechnology, 23, 9, 1105-1116 (2005).

Also encompassed within the present invention are methods for preparingand generating multiparatopic (such as e.g. biparatopic, triparatopic,etc.) amino acids of the invention.

Without being limiting, a method for preparing and generatingbiparatopic amino acids of the invention may comprise at least the stepsof:

-   a) providing a nucleic acid sequence encoding an HER2 binding amino    acid sequence fused to a set, collection or library of nucleic acid    sequences encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for an antigenic    determinant on HER2 different from the antigenic determinant    recognized by the HER2 binding amino acid sequence;    and-   c) isolating the nucleic acid sequence encoding an HER2 binding    amino acid sequence fused to the nucleic acid sequence obtained in    b), followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences andagain screened for nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for an antigenicdeterminant on HER2 different from the antigenic determinant of the HER2binding amino acid sequence and the antigenic determinant of b) in orderto obtain a triparatopic or multiparatopic amino acid sequencerespectively.

in such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 bindingamino acid sequence fused to the set, collection or library ofnucleotide sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

According to a particularly preferred aspect, a method for preparing andgenerating biparatopic amino acids of the invention may comprise atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,    in which each nucleic acid sequence in said set, collection or    library encodes a fusion protein that comprises a first amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 that is fused    (optionally via a linker sequence) to a second amino acid sequence,    in which essentially each second amino acid sequence (or most of    these) is a different member of a set, collection or library of    different amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2;    and-   c) isolating the nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2,    obtained in b), optionally followed by expressing the encoded amino    acid sequence.

In this preferred method, the first amino acid sequence in the fusionprotein encoded by said set collection or library of nucleic acidsequences may be the same amino acid sequence for all members of theset, collection or library of nucleic acid sequences encoding the fusionprotein; or the first amino acid sequence in the fusion protein encodedby said set collection or library of nucleic acid sequences may also bea member of a set collection or library of different amino acidsequences.

Again, in such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences that form part of the fusionprotein may for example be a set, collection or library of nucleic acidsequences encoding a naïve set, collection or library of immunoglobulinsequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofimmunoglobulin sequences; and/or a set, collection or library of nucleicacid sequences encoding a set, collection or library of immunoglobulinsequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 bindingamino acid sequence fused to the set, collection or library ofnucleotide sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 11.05-1116 (2005).

In step b), the set, collection or library of nucleic acid sequences mayalso be screened for nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for both the firstantigenic determinant, part, domain or epitope on HER2 and the secondantigenic determinant, part, domain or epitope on HER2. This may forexample be performed in a subsequent steps (i.e. by in a first stepscreening or selecting for nucleic acid sequences that encode an aminoacid sequence that can bind to and/or has affinity for the secondantigenic determinant, part, domain or epitope on HER2, and subsequentlyin a second step selecting or screening for nucleic acid sequences thatencode an amino acid sequence that can bind to and/or has affinity forthe first antigenic determinant, part, domain or epitope on HER2; orvisa versa) or in a single step (i.e. by simultaneously screening orselecting for nucleic acid sequences that encode an amino acid sequencethat can bind to and/or has affinity for both the first antigenicdeterminant, part, domain or epitope on HER2 and the second antigenicdeterminant, part, domain or epitope on HER2).

In a preferred aspect of the above method, the first amino acid sequenceused in step a) is preferably such that (i) it can bind to and/or hasaffinity for the Herceptin® binding site on HER2 (and may in particularbe directed against domain IV of HER2, more in particular the C-terminusof domain IV of HER2) and/or (ii) competes with Herceptin® for bindingto HER-2; and in step b), the set, collection or library of nucleic acidsequences is screened for nucleic acid sequences that encode (i) anamino acid sequence that can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) an aminoacid sequence that can compete with Omnitarg (or the Omnitarg Fab usedin Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) ispreferably such that (i) it can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain IT of HER2,more in particular the middle of domain II of HER2) and/or (ii) competeswith Omnitarg for binding to HER-2; and in step b), the set, collectionor library of nucleic acid sequences is screened for nucleic acidsequences that encode (i) an amino acid sequence that can bind to and/orhas affinity for the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or (ii) an amino acid sequence that can compete withHerceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

It is also possible, in step b), to screen for nucleic acid sequencesthat both (i) encode an amino acid sequence that can bind to and/or hasaffinity for the Omnitarg binding site on HER2 (and in particular domainII of HER2, more in particular the middle of domain II of HER2) and/orthat can compete with Omnitarg (or the Omnitarg Fab used in Example 9)for binding to HER-2; and that also (ii) encode an amino acid sequencethat can bind to and/or has affinity for the Herceptin® binding site onHER2 (and in particular domain IV of HER2, more in particular theC-terminus of domain IV of HER2) and/or that can compete with Herceptin®for binding to HER-2. Again, this may be performed in separate steps ora single step, and by selecting or screening in the presence ofHerceptin® and/or Omnitarg, as applicable.

It will also be clear to the skilled person that the above methods maybe performed by screening a set, collection or library of amino acidsequences that correspond to (e.g. are encoded by) the nucleic acidsequences used in the above method; and such methods form furtheraspects of the invention.

The invention in a further aspect provides a method for preparing andgenerating biparatopic amino acids of the invention which comprises atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,    in which each nucleic acid sequence in said set, collection or    library encodes a fusion protein that comprises a first amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 that is fused via a    linker sequence to a second amino acid sequence that can bind to    and/or has affinity for a second antigenic determinant, part, domain    or epitope on HER2 (which may be the same or different as the first    antigenic determinant, part, domain or epitope on HER2), in which    essentially each nucleic acid sequence (or most of these) encodes a    fusion protein with a different linker sequence so as to provide a    set, collection or library of nucleic acid sequences encoding    different fusion proteins;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for the first and    second antigenic determinant, part, domain or epitope on HER2;    and-   c) isolating the nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for the first and    second antigenic determinant, part, domain or epitope on HER2,    optionally followed by expressing the encoded amino acid sequence.

As will be clear to the skilled person, this method can be used toscreen for suitable or even optimal linker lengths for linking the firstand second amino acid sequence. For example, in this aspect, the firstamino acid sequence may be an amino acid sequence that can bind toand/or has affinity for the Omnitarg binding site on HER2 (and may inparticular domain II of HER2, more in particular the middle of domain IIof HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab usedin Example 9); and the second amino acid sequence may be an amino acidsequence that can bind to and/or has affinity for the Herceptin® bindingsite on HER2 (and in particular domain IV of HER2, more in particularthe C-terminus of domain IV of HER2) and/or that can compete withHerceptin® for binding to HER-2 (or visa versa). The screening andselection step b) may be performed as further described above.

Another method for preparing and generating biparatopic amino acids ofthe invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for a set, collection or library of nucleic acid sequences    that encode an amino acid sequence that can bind to and/or has    affinity for HER2;-   c) ligating said set, collection or library of nucleic acid    sequences that encode an amino acid sequence that can bind to and/or    has affinity for HER2 to another nucleic acid sequence that encodes    an amino acid sequence that can bind to and/or has affinity for HER2    (e.g. a nucleic acid sequence that encodes an amino acid sequence    that competes with Herceptin® for binding HER2);    and-   d) from the set, collection or library of nucleic acid sequences    obtained in c), isolating the nucleic acid sequences encoding a    biparatopic amino acid sequence that can bind to and/or has affinity    for HER2 (and e.g. further selecting for nucleic acid sequences that    encode a biparatopic amino acid sequence that antagonizes with    higher potency compared to the monovalent amino acid sequences),    followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences thatcan bind to and/or have affinity for HER2 in order to obtain atriparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 can beobtained by any selection or screening method known in the art for theselection and/or screening of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 andas, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on aphage, phagemid, ribosome or suitable micro-organism (such as yeast),such as to facilitate screening. Suitable methods, techniques and hostorganisms for displaying and screening (a set, collection or library of)nucleotide sequences encoding amino acid sequences will be clear to theperson skilled in the art, for example on the basis of the furtherdisclosure herein. Reference is also made to the review by Hoogenboom inNature Biotechnology, 23, 9, 1105-1116 (2005).

Another method for preparing and generating biparatopic amino acids ofthe invention may comprise at least the steps of:

-   a) providing a first set, collection or library of nucleic acid    sequences encoding amino acid sequences;-   b) screening said first set, collection or library of nucleic acid    sequences for a nucleic acid sequence that encodes an amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2;-   c) ligating the nucleic acid sequence encoding said amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 obtained in b) to    another set, collection or library of nucleic acid sequences    encoding amino acid sequences to obtain a set, collection or library    of nucleic acid sequences that encode fusion proteins;-   d) screening said set, collection or library of nucleic acid    sequences obtained in step c) for a nucleic acid sequence that    encodes an amino acid sequence that can bind a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2;    and-   e) isolating the nucleic acid sequence that encodes an amino acid    sequence that can bind to and/or has affinity for the first and    second antigenic determinant, part, domain or epitope on HER2,    optionally followed by expressing the encoded amino acid sequence.

In a preferred aspect of the above method, the first amino acid sequenceobtained in step b) is preferably such that (i) it can bind to and/orhas affinity for the Herceptin® binding site on HER2 (and may inparticular be directed against domain IV of HER2, more in particular theC-terminus of domain IV of HER2) and/or (ii) competes with Herceptin®for binding to HER-2; and in step d), the set, collection or library ofnucleic acid sequences is screened for nucleic acid sequences thatencode (i) an amino acid sequence that can bind to and/or has affinityfor the Omnitarg binding site on HER2 (and may in particular domain IIof HER2, more in particular the middle of domain II of HER2) and/or (ii)an amino acid sequence that can compete with Omnitarg (or the OmnitargFab used in Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence obtained in step b) ispreferably such that (i) it can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) competeswith Omnitarg for binding to HER-2; and in step d), the set, collectionor library of nucleic acid sequences is screened for nucleic acidsequences that encode (i) an amino acid sequence that can bind to and/orhas affinity for the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or (ii) an amino acid sequence that can compete withHerceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

It is also possible, in step d), to screen for nucleic acid sequencesthat both (i) encode an amino acid sequence that can bind to and/or hasaffinity for the Omnitarg binding site on HER2 (and in particular domainII of HER2, more in particular the middle of domain II of HER2) and/orthat can compete with Omnitarg (or the Omnitarg Fab used in Example 9)for binding to HER-2; and that also (ii) encode an amino acid sequencethat can bind to and/or has affinity for the Herceptin® binding site onHER2 (and in particular domain IV of HER2, more in particular theC-terminus of domain IV of HER2) and/or that can compete with Herceptin®for binding to HER-2. Again, this may be performed in separate steps ora single step, and by selecting or screening in the presence ofHerceptin® and/or Omnitarg, as applicable.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences thatcan bind to and/or have affinity for HER2 in order to obtain atriparatopic or multiparatopic amino acid sequence respectively. In sucha method, the set, collection or library of nucleic acid sequencesencoding amino acid sequences may for example be a set, collection orlibrary of nucleic acid sequences encoding a naïve set, collection orlibrary of immunoglobulin sequences; a set, collection or library ofnucleic acid sequences encoding a synthetic or semi-synthetic set,collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 can beobtained by any selection or screening method known in the art for theselection and/or screening of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 andas, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on aphage, phagemid, ribosome or suitable micro-organism (such as yeast),such as to facilitate screening. Suitable methods, techniques and hostorganisms for displaying and screening (a set, collection or library of)nucleotide sequences encoding amino acid sequences will be clear to theperson skilled in the art, for example on the basis of the furtherdisclosure herein. Reference is also made to the review by Hoogenboom inNature Biotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises one ofthe above methods and in addition at least the steps of determining thenucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Another method for preparing multivalent and/ormultiparatopic/biparatopic amino acids or constructs of the inventionmay comprise at least the steps of linking two or more monovalent aminoacid sequences or monovalent construct of the invention and for exampleone or more linkers together in a suitable manner. The monovalentconstructs (and linkers) can be coupled by any method known in the artand as further described herein. Preferred techniques include thelinking of the nucleic acid sequences that encode the monovalentconstructs (and linkers) to prepare a genetic construct that expressesthe multivalent and/or multiparatopic/biparatopic amino acid orconstruct. Techniques for linking amino acid sequences or nucleic acidsequences will be clear to the skilled person, and reference is againmade to the standard handbooks, such as Sambrook et al. and Ausubel etal., mentioned above, as well as the Examples below.

Accordingly, the present invention also relates to the use of amonovalent construct (which may comprise or essentially consists of anamino acid sequence of the invention such as a domain antibody, an aminoacid sequence that is suitable for use as a domain antibody, a singledomain antibody, an amino acid sequence that is suitable for use as asingle domain antibody, a “dAb”, an amino acid sequences that issuitable for use as a dAb, or a Nanobody) in providing and/or preparinga multivalent (such as multiparatopic, and preferably biparatopic)compound or construct. The monovalent construct is then used as abinding domain or binding unit in providing and/or preparing themultivalent (such as multiparatopic, and preferably biparatopic)construct comprising two (e.g. in a biparatopic construct) or more (e.g.in a multiparatopic construct) binding units. In this respect, themonovalent construct may be used as a binding domain or binding unit inproviding and/or preparing a multivalent (such as multiparatopic, andpreferably biparatopic) construct of the invention comprising two ormore binding units.

In a preferred aspect, the monovalent construct (which may comprise oressentially consists of an amino acid sequence of the invention such asa domain antibody, an amino acid sequence that is suitable for use as adomain antibody, a single domain antibody, an amino acid sequence thatis suitable for use as a single domain antibody, a “dAb”, an amino acidsequences that is suitable for use as a dAb, or a Nanobody) is used inproviding and/or preparing a multivalent (such as multiparatopic, andpreferably biparatopic) construct that exhibits intramolecular bindingcompared to intermolecular binding. In such multivalent constructs ofthe invention that comprises amino acid sequences directed against twoor more (different) antigenic determinants on the same antigen (forexample against different epitopes of an antigen and/or againstdifferent subunits of a multimeric receptor or protein), the length andflexibility of the linker are preferably such that, when the multivalentconstruct binds to HER-2, at least two and preferably all of the aminoacid sequences that are present in the multivalent construct can(simultaneously) bind to each of their intended antigenic determinants,epitopes, parts or domains, most preferably so as to allow binding withincreased avidity and also intramolecular binding and/or recognition.Accordingly, the present invention also relates to the use of amonovalent construct (which may comprise or essentially consists of anamino acid sequence of the invention such as a domain antibody, an aminoacid sequence that is suitable for use as a domain antibody, a singledomain antibody, an amino acid sequence that is suitable for use as asingle domain antibody, a “dAb”, an amino acid sequences that issuitable for use as a dAb, or a Nanobody) as a binding domain or bindingunit in providing and/or preparing a multivalent (such asmultiparatopic, and preferably biparatopic) construct, wherein thebinding domains or binding units are linked via a linker such that themultivalent (such as multiparatopic, and preferably biparatopic)construct preferably exhibits intramolecular binding compared tointermolecular binding.

In some of the most preferred multiparatopic polypeptides of theinvention, (i) at least one monovalent construct of the invention (andin particular at least one Nanobody) is used that is directed againstthe Omnitarg binding site on HER2 (and in particular against domain IIof HER2, and more in particular against the middle of domain II of HER2)and/or that is capable of competing with Omnitarg for binding to HER-2;and at least one amino acid sequence of the invention (and in particularat least one Nanobody) is used that is directed against anotherantigenic determinant, epitope, part or domain of HER2. In such apreferred multiparatopic construct of the invention, the linker is mostpreferably such that the multiparatopic construct of the invention iscapable of (simultaneously) binding to both the Omnitarg binding site onHER2 (and in particular against domain II of HER2, and more inparticular against the middle of domain II of HER2) as well as the otherantigenic determinant, epitope, part or domain of HER2, again mostpreferably so as to allow binding with increased avidity and alsointramolecular binding and/or recognition. Accordingly, also encompassedin the present invention is the use of a monovalent construct comprisingan amino acid of the invention (and in particular a Nanobody) that isdirected against the Omnitarg binding site on HER2 (and in particularagainst domain II of HER2, and more in particular against the middle ofdomain II of HER2) and/or that is capable of competing with Omnitarg forbinding to HER-2, as a binding domain or binding unit in providingand/or preparing a multiparatopic (such as biparatopic) construct,wherein the binding domains or binding units are linked via a linkersuch that the multiparatopic (such as biparatopic) construct preferablyexhibits intramolecular binding compared to intermolecular binding.

In some of the most preferred multiparatopic polypeptides of theinvention, (i) at least one monovalent construct of the invention (andin particular at least one Nanobody) is used that is directed againstthe Herceptin® binding site on HER2 (and in particular against domain IVof HER2, and more in particular against the C-terminus of domain IV ofHER2) and/or that is capable of competing with Herceptin® for binding toHER-2; and at least one amino acid of the invention (and in particularat least one Nanobody) is used that is directed against anotherantigenic determinant, epitope, part or domain of HER2. In such apreferred multiparatopic construct of the invention, the linker is mostpreferably such that the multiparatopic construct of the invention iscapable of (simultaneously) binding to both the Herceptin® binding siteon HER2 (and in particular against domain IV of HER2, and more inparticular against the C-terminus of domain IV of HER2), as well as theother antigenic determinant, epitope, part or domain of HER2, again mostpreferably so as to allow binding with increased avidity and alsointramolecular binding and/or recognition. Accordingly, also encompassedin the present invention is the use of a monovalent construct comprisingan amino acid sequence of the invention (and in particular at least oneNanobody) that is directed against the Herceptin® binding site on HER2(and in particular against domain IV of HER2, and more in particularagainst the C-terminus of domain IV of HER2) and/or that is capable ofcompeting with Herceptin® for binding to HER-2, as a binding domain orbinding unit in providing and/or preparing a multiparatopic (such as abiparatopic) construct, wherein the binding domains or binding units arelinked via a linker such that the multiparatopic (such as biparatopic)construct preferably exhibits intramolecular binding compared tointermolecular binding.

In some of the most preferred multiparatopic polypeptides of theinvention, (i) at least one monovalent construct of the invention (andin particular at least one Nanobody) is used that is directed againstthe Omnitarg binding site on HER2 (and in particular against domain IIof HER2, and more in particular against the middle of domain II of HER2)and/or that is capable of competing with Omnitarg for binding to HER-2;and at least one monovalent construct of the invention (and inparticular at least one Nanobody) is used that is directed against theHerceptin® binding site on HER2 (and in particular against domain IV ofHER2, and more in particular against the C-terminus of domain IV ofHER2) and/or that is capable of competing with Herceptin® for binding toHER-2. In such a preferred multiparatopic construct of the invention,the linker is most preferably such that the multiparatopic construct ofthe invention is capable of (simultaneously) binding to both theOmnitarg binding site on HER2 (and in particular against domain II ofHER2, and more in particular against the middle of domain II of HER2) aswell as the Herceptin® binding site on HER2 (and in particular againstdomain IV of HER2, and more in particular against the C-terminus ofdomain IV of HER2), again most preferably so as to allow binding withincreased avidity and also intramolecular binding and/or recognition.Accordingly, also encompassed in the present invention is the use of amonovalent construct comprising an amino acid sequence of the invention(and in particular at least one Nanobody) that is directed against theHerceptin® binding site on HER2 (and in particular against domain IV ofHER2, and more in particular against the C-terminus of domain IV ofHER2) and/or that is capable of competing with Herceptin® for binding toHER-2, and a monovalent construct comprising an amino acid of theinvention (and in particular a Nanobody) that is directed against theOmnitarg binding site on HER2 (and in particular against domain II ofHER2, and more in particular against the middle of domain II of HER2)and/or that is capable of competing with Omnitarg for binding to HER-2,as binding domains or binding units in providing and/or preparing amultiparatopic (such as a biparatopic) construct, wherein the bindingdomains or binding units are linked via a linker such that themultiparatopic (such as biparatopic) construct preferably exhibitsintramolecular binding compared to intermolecular binding.

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises one ofthe above methods and in addition at least the steps of determining thenucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

In this respect, the present invention also relates to the use of anucleic acid or nucleotide sequence that encodes a monovalent constructof the invention for the preparation of a genetic construct (as furtherdefined herein) that encodes a multivalent (such as multiparatopic, andpreferably biparatopic) construct. Also, as will be clear to the skilledperson, to prepare such a genetic construct, encoding a multivalent(such as multiparatopic, and preferably biparatopic) construct of theinvention, several nucleotide sequences, such as at least two nucleotidesequences encoding a monovalent construct of the invention and forexample nucleic acids encoding one or more linkers can be linkedtogether in a suitable manner. Such genetic constructs generally alsocomprises one or more elements of genetic constructs known per se, suchas for example one or more suitable regulatory elements (such as asuitable promoter(s), enhancer(s), terminator(s), etc.) and the furtherelements of genetic constructs referred to herein.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned above,as well as the Examples below.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with HER2. Some preferred but non-limiting applications anduses will become clear from the further description herein.

The invention also relates to the amino acid sequences, compounds,constructs, polypeptides, nucleic acids, host cells, products andcompositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder that can be prevented or treated by administering, to a subjectin need thereof, of (a pharmaceutically effective amount of) an aminoacid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of cancers and/ortumors.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks, such as    Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),    Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et    al, eds., “Current protocols in molecular biology”, Green Publishing    and Wiley Interscience, New York (1987); Lewin, “Genes II”, John    Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of    Gene Manipulation: An Introduction to Genetic Engineering”, 2nd    edition, University of California Press, Berkeley, Calif. (1981);    Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh    (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.    Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”    (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.    (2005), as well as to the general background art cited herein;-   b) Unless indicated otherwise, the term “immunoglobulin    sequence”—whether used herein to refer to a heavy chain antibody or    to a conventional 4-chain antibody—is used as a general term to    include both the full-size antibody, the individual chains thereof,    as well as all parts, domains or fragments thereof (including but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H)/V_(L) domains, respectively). In addition, the term    “sequence” as used herein (for example in terms like “immunoglobulin    sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)    sequence” or “protein sequence”), should generally be understood to    include both the relevant amino acid sequence as well as nucleic    acids or nucleotide sequences encoding the same, unless the context    requires a more limited interpretation. Also, the term “nucleotide    sequence” as used herein also encompasses a nucleic acid molecule    with said nucleotide sequence, so that the terms “nucleotide    sequence” and “nucleic acid” should be considered equivalent and are    used interchangeably herein;-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table    A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residu can generallybe considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated by    dividing [the number of nucleotides in the first nucleotide sequence    that are identical to the nucleotides at the corresponding positions    in the second nucleotide sequence] by [the total number of    nucleotides in the first nucleotide sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of a nucleotide in the second nucleotide sequence—compared to the    first nucleotide sequence—is considered as a difference at a single    nucleotide (position).-    Alternatively, the degree of sequence identity between two or more    nucleotide sequences may be calculated using a known computer    algorithm for sequence alignment such as NCBI Blast v2.0, using    standard settings.-    Some other techniques, computer algorithms and settings for    determining the degree of sequence identity are for example    described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318,    WO 00/78972, WO 98/49185 and GB 2 357 768-A.-    Usually, for the purpose of determining the percentage of “sequence    identity” between two nucleotide sequences in accordance with the    calculation method outlined hereinabove, the nucleotide sequence    with the greatest number of nucleotides will be taken as the “first”    nucleotide sequence, and the other nucleotide sequence will be taken    as the “second” nucleotide sequence;-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.-    Alternatively, the degree of sequence identity between two amino    acid sequences may be calculated using a known computer algorithm,    such as those mentioned above for determining the degree of sequence    identity for nucleotide sequences, again using standard settings.-    Usually, for the purpose of determining the percentage of “sequence    identity” between two amino acid sequences in accordance with the    calculation method outlined hereinabove, the amino acid sequence    with the greatest number of amino acid residues will be taken as the    “first” amino acid sequence, and the other amino acid sequence will    be taken as the “second” amino acid sequence.-    Also, in determining the degree of sequence identity between two    amino acid sequences, the skilled person may take into account    so-called “conservative” amino acid substitutions, which can    generally be described as amino acid substitutions in which an amino    acid residue is replaced with another amino acid residue of similar    chemical structure and which has little or essentially no influence    on the function, activity or other biological properties of the    polypeptide. Such conservative amino acid substitutions are well    known in the art, for example from WO 04/037999, GB-A-3 357 768, WO    98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or    combinations of such substitutions may be selected on the basis of    the pertinent teachings from WO 04/037999 as well as WO 98/49185 and    from the further references cited therein.-    Such conservative substitutions preferably are substitutions in    which one amino acid within the following groups (a)-(e) is    substituted by another amino acid residue within the same group: (a)    small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,    Pro and Gly; (b) polar, negatively charged residues and their    (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively    charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar    residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:    Phe, Tyr and Trp.-    Particularly preferred conservative substitutions are as follows:    Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His;    Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala    or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu    into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into    Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser    into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into    Val, into Ile or into Leu.-    Any amino acid substitutions applied to the polypeptides described    herein may also be based on the analysis of the frequencies of amino    acid variations between homologous proteins of different species    developed by Schulz et al., Principles of Protein Structure,    Springer-Verlag, 1978, on the analyses of structure forming    potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974    and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of    hydrophobicity patterns in proteins developed by Eisenberg et al.,    Proc. Nad. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J    Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev.    Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their    entirety by reference. Information on the primary, secondary and    tertiary structure of Nanobodies is given in the description herein    and in the general background art cited above. Also, for this    purpose, the crystal structure of a V_(HH) domain from a llama is    for example given by Desmyter et al., Nature Structural. Biology,    Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology    (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361    (1999). Further information about some of the amino acid residues    that in conventional V_(H) domains form the V_(H)/V_(L) interface    and potential camelizing substitutions on these positions can be    found in the prior art cited above.-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this may mean that the latter    nucleotide sequence or amino acid sequence has been incorporated    into the firstmentioned nucleotide sequence or amino acid sequence,    respectively, but more usually this generally means that the    firstmentioned nucleotide sequence or amino acid sequence comprises    within its sequence a stretch of nucleotides or amino acid residues,    respectively, that has the same nucleotide sequence or amino acid    sequence, respectively, as the latter sequence, irrespective of how    the firstmentioned sequence has actually been generated or obtained    (which may for example be by any suitable method described herein).    By means of a non-limiting example, when a Nanobody of the invention    is said to comprise a CDR sequence, this may mean that said CDR    sequence has been incorporated into the Nanobody of the invention,    but more usually this generally means that the Nanobody of the    invention contains within its sequence a stretch of amino acid    residues with the same amino acid sequence as said CDR sequence,    irrespective of how said Nanobody of the invention has been    generated or obtained. It should also be noted that when the latter    amino acid sequence has a specific biological or structural    function, it preferably has essentially the same, a similar or an    equivalent biological or structural function in the firstmentioned    amino acid sequence (in other words, the firstmentioned amino acid    sequence is preferably such that the latter sequence is capable of    performing essentially the same, a similar or an equivalent    biological or structural function). For example, when a Nanobody of    the invention is said to comprise a CDR sequence or framework    sequence, respectively, the CDR sequence and framework are    preferably capable, in said Nanobody, of functioning as a CDR    sequence or framework sequence, respectively. Also, when a    nucleotide sequence is said to comprise another nucleotide sequence,    the firstmentioned nucleotide sequence is preferably such that, when    it is expressed into an expression product (e.g. a polypeptide), the    amino acid sequence encoded by the latter nucleotide sequence forms    part of said expression product (in other words, that the latter    nucleotide sequence is in the same reading frame as the    firstmentioned, larger nucleotide sequence).-   j) A nucleic acid sequence or amino acid sequence is considered to    be “(in) essentially isolated (form)”—for example, compared to its    native biological source and/or the reaction medium or cultivation    medium from which it has been obtained—when it has been separated    from at least one other component with which it is usually    associated in said source or medium, such as another nucleic acid,    another protein/polypeptide, another biological component or    macromolecule or at least one contaminant, impurity or minor    component. In particular, a nucleic acid sequence or amino acid    sequence is considered “essentially isolated” when it has been    purified at least 2-fold, in particular at least 10-fold, more in    particular at least 100-fold, and up to 1000-fold or more. A nucleic    acid sequence or amino acid sequence that is “in essentially    isolated form” is preferably essentially homogeneous, as determined    using a suitable technique, such as a suitable chromatographical    technique, such as polyacrylamide-gel electrophoresis;-   k) The term “domain” as used herein generally refers to a globular    region of an amino acid sequence (such as an antibody chain, and in    particular to a globular region of a heavy chain antibody), or to a    polypeptide that essentially consists of such a globular region.    Usually, such a domain will comprise peptide loops (for example 3 or    4 peptide loops) stabilized, for example, as a sheet or by disulfide    bonds. The term “binding domain” refers to such a domain that is    directed against an antigenic determinant (as defined herein);-   l) The term “antigenic determinant” refers to the epitope on the    antigen recognized by the antigen-binding molecule (such as a    Nanobody or a polypeptide of the invention) and more in particular    by the antigen-binding site of said molecule. The terms “antigenic    determinant” and “epitope” may also be used interchangeably herein.-   m) An amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity. The affinity, represented by the    equilibrium constant for the dissociation of an antigen with an    antigen-binding protein (K_(D)), is a measure for the binding    strength between an antigenic determinant and an antigen-binding    site on the antigen-binding protein: the lesser the value of the    K_(D), the stronger the binding strength between an antigenic    determinant and the antigen-binding molecule (alternatively, the    affinity can also be expressed as the affinity constant (K_(A)),    which is 1/K_(D)). As will be clear to the skilled person (for    example on the basis of the further disclosure herein), affinity can    be determined in a manner known per se, depending on the specific    antigen of interest. Avidity is the measure of the strength of    binding between an antigen-binding molecule (such as a Nanobody or    polypeptide of the invention) and the pertinent antigen. Avidity is    related to both the affinity between an antigenic determinant and    its antigen binding site on the antigen-binding molecule and the    number of pertinent binding sites present on the antigen-binding    molecule. Typically, antigen-binding proteins (such as the amino    acid sequences, Nanobodies and/or polypeptides of the invention)    will bind to their antigen with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter    (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²    liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more    and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, a monovalent immunoglobulin sequence of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein.-    The dissociation constant may be the actual or apparent    dissociation constant, as will be clear to the skilled person.    Methods for determining the dissociation constant will be clear to    the skilled person, and for example include the techniques mentioned    herein. In this respect, it will also be clear that it may not be    possible to measure dissociation constants of more then 10⁻⁴    moles/liter or 10⁻³ moles/liter (e.g. of 10⁻² moles/liter).    Optionally, as will also be clear to the skilled person, the (actual    or apparent) dissociation constant may be calculated on the basis of    the (actual or apparent) association constant (K_(A)), by means of    the relationship [K_(D)=1/K_(A)].-    The affinity denotes the strength or stability of a molecular    interaction. The affinity is commonly given as by the K_(D), or    dissociation constant, which has units of mol/liter (or M). The    affinity can also be expressed as an association constant, K_(A),    which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the    present specification, the stability of the interaction between two    molecules (such as an amino acid sequence, Nanobody or polypeptide    of the invention and its intended target) will mainly be expressed    in terms of the K_(D) value of their interaction; it being clear to    the skilled person that in view of the relation K_(A)=1/K_(D),    specifying the strength of molecular interaction by its K_(D) value    can also be used to calculate the corresponding K_(A) value. The    K_(D)-value characterizes the strength of a molecular interaction    also in a thermodynamic sense as it is related to the free energy    (DG) of binding by the well known relation DG=RT·ln(K_(D))    (equivalently DG=−RT·ln(K_(A))), where R equals the gas constant, T    equals the absolute temperature and ln denotes the natural    logarithm.-    The K_(D) for biological interactions which are considered    meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M (0.1    nM) to 10⁻⁵M (10000 nM). The stronger an interaction is, the lower    is its K_(D).-    The K_(D) can also be expressed as the ratio of the dissociation    rate constant of a complex, denoted as k_(off), to the rate of its    association, denoted k_(on) (so that K_(D)=k_(off)/k_(on) and    K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s    is the SI unit notation of second). The on-rate k_(on) has units M⁻¹    s⁻¹. The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,    approaching the diffusion-limited association rate constant for    bimolecular interactions. The off-rate is related to the half-life    of a given molecular interaction by the relation    t_(1/2)=ln(2)/k_(off). The off-rate may vary between 10⁻⁶ s⁻¹ (near    irreversible complex with a t_(1/2) of multiple days) to 1 s⁻¹    (t_(1/2)=0.69 s).-    The affinity of a molecular interaction between two molecules can    be measured via different techniques known per se, such as the well    known surface plasmon resonance (SPR) biosensor technique (see for    example Ober et al., Intern. Immunology, 13, 1551-1559, 2001) where    one molecule is immobilized on the biosensor chip and the other    molecule is passed over the immobilized molecule under flow    conditions yielding k_(on), k_(off) measurements and hence K_(D) (or    K_(A)) values. This can for example be performed using the    well-known BIACORE instruments.-    It will also be clear to the skilled person that the measured K_(D)    may correspond to the apparent K_(D) if the measuring process    somehow influences the intrinsic binding affinity of the implied    molecules for example by artefacts related to the coating on the    biosensor of one molecule. Also, an apparent K_(D) may be measured    if one molecule contains more than one recognition sites for the    other molecule. In such situation the measured affinity may be    affected by the avidity of the interaction by the two molecules.-    Another approach that may be used to assess affinity is the 2-step    ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et    al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes    a solution phase binding equilibrium measurement and avoids possible    artefacts relating to adsorption of one of the molecules on a    support such as plastic.-    However, the accurate measurement of K_(D) may be quite    labor-intensive and as consequence, often apparent K_(D) values are    determined to assess the binding strength of two molecules. It    should be noted that as long all measurements are made in a    consistent way (e.g. keeping the assay conditions unchanged)    apparent K_(D) measurements can be used as an approximation of the    true K_(D) and hence in the present document K_(D) and apparent    K_(D) should be treated with equal importance or relevance. Finally,    it should be noted that in many situations the experienced scientist    may judge it to be convenient to determine the binding affinity    relative to some reference molecule. For example, to assess the    binding strength between molecules A and B, one may e.g. use a    reference molecule C that is known to bind to B and that is suitably    labelled with a fluorophore or chromophore group or other chemical    moiety, such as biotin for easy detection in an ELISA or FACS    (Fluorescent activated cell sorting) or other format (the    fluorophore for fluorescence detection, the chromophore for light    absorption detection, the biotin for streptavidin-mediated ELISA    detection). Typically, the reference molecule C is kept at a fixed    concentration and the concentration of A is varied for a given    concentration or amount of B. As a result an IC₅₀ value is obtained    corresponding to the concentration of A at which the signal measured    for C in absence of A is halved. Provided K_(D ref), the K_(D) of    the reference molecule, is known, as well as the total concentration    c_(ref) of the reference molecule, the apparent K_(D) for the    interaction A-B can be obtained from following formula:    K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if c_(ref)<<K_(D ref),    K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in a    consistent way (e.g. keeping c_(ref) fixed) for the binders that are    compared, the strength or stability of a molecular interaction can    be assessed by the IC₅₀ and this measurement is judged as equivalent    to K_(D) or to apparent K_(D) throughout this text.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomologus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as to the standard handbooks, such as Kenneth, A et    al: Chemical Stability of Pharmaceuticals: A Handbook for    Pharmacists and Peters et al, Pharmacokinete analysis: A Practical    Approach (1996). Reference is also made to “Pharmacokinetics”, M    Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition    (1982).-    As will also be clear to the skilled person (see for example pages    6 and 7 of WO 04/003019 and in the further references cited    therein), the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC). In the    present specification, an “increase in half-life” refers to an    increase in any one of these parameters, such as any two of these    parameters, or essentially all three these parameters. As used    herein “increase in half-life” or “increased half-life” in    particular refers to an increase in the t1/2-beta, either with or    without an increase in the t1/2-alpha and/or the AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.-    As will be clear to the skilled person, “modulating” may also    involve effecting a change (which may either be an increase or a    decrease) in affinity, avidity, specificity and/or selectivity of a    target or antigen for one or more of its ligands, binding partners,    partners for association into a homomultimeric or heteromultimeric    form, or substrates; and/or effecting a change (which may either be    an increase or a decrease) in the sensitivity of the target or    antigen for one or more conditions in the medium or surroundings in    which the target or antigen is present (such as pH, ion strength,    the presence of co-factors, etc.), compared to the same conditions    but without the presence of the construct of the invention. As will    be clear to the skilled person, this may again be determined in any    suitable manner and/or using any suitable assay known per se,    depending on the target or antigen involved.-    “Modulating” may also mean effecting a change (i.e. an activity as    an agonist, as an antagonist or as a reverse agonist, respectively,    depending on the target or antigen and the desired biological or    physiological effect) with respect to one or more biological or    physiological mechanisms, effects, responses, functions, pathways or    activities in which the target or antigen (or in which its    substrate(s), ligand(s) or pathway(s) are involved, such as its    signalling pathway or metabolic pathway and their associated    biological or physiological effects) is involved. Again, as will be    clear to the skilled person, such an action as an agonist or an    antagonist may be determined in any suitable manner and/or using any    suitable (in vitro and usually cellular or in assay) assay known per    se, depending on the target or antigen involved. In particular, an    action as an agonist or antagonist may be such that an intended    biological or physiological activity is increased or decreased,    respectively, by at least 1%, preferably at least 5%, such as at    least 10% or at least 25%, for example by at least 50%, at least    60%, at least 70%, at least 80%, or 90% or more, compared to the    biological or physiological activity in the same assay under the    same conditions but without the presence of the construct of the    invention.-    Modulating may for example also involve allosteric modulation of    the target or antigen; and/or reducing or inhibiting the binding of    the target or antigen to one of its substrates or ligands and/or    competing with a natural ligand, substrate for binding to the target    or antigen. Modulating may also involve activating the target or    antigen or the mechanism or pathway in which it is involved.    Modulating may for example also involve effecting a change in    respect of the folding or confirmation of the target or antigen, or    in respect of the ability of the target or antigen to fold, to    change its confirmation (for example, upon binding of a ligand), to    associate with other (sub)units, or to disassociate.-    Modulating may for example also involve effecting a change in the    ability of the target or antigen to transport other compounds or to    serve as a channel for other compounds (such as ions).-    Modulating may be reversible or irreversible, but for    pharmaceutical and pharmacological purposes will usually be in a    reversible manner.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10.000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10.000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a Nanobody, polypeptide or    compound or construct of the invention) to interfere with the    binding of other amino acid sequences or binding agents of the    invention to a given target. The extend to which an amino acid    sequence or other binding agents of the invention is able to    interfere with the binding of another to HER2, and therefore whether    it can be said to cross-block according to the invention, can be    determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a Biacore instrument    which can measure the extent of interactions using surface plasmon    resonance technology. Another suitable quantitative cross-blocking    assay uses an ELISA-based approach to measure competition between    amino acid sequences or other binding agents in terms of their    binding to the target. The following generally describes a suitable    Biacore assay for determining whether an amino acid sequence or    other binding agent cross-blocks or is capable of cross-blocking    according to the invention. It will be appreciated that the assay    can be used with any of the amino acid sequences or other binding    agents described herein. The Biacore machine (for example the    Biacore 3000) is operated in line with the manufacturer's    recommendations. Thus in one cross-blocking assay, the target    protein is coupled to a CM5 Biacore chip using standard amine    coupling chemistry to generate a surface that is coated with the    target. Typically 200-800 resonance units of the target would be    coupled to the chip (an amount that gives easily measurable levels    of binding but that is readily saturable by the concentrations of    test reagent being used). Two test amino acid sequences (termed A*    and B*) to be assessed for their ability to cross-block each other    are mixed at a one to one molar ratio of binding sites in a suitable    buffer to create the test mixture. When calculating the    concentrations on a binding site basis the molecular weight of an    amino acid sequence is assumed to be the total molecular weight of    the amino acid sequence divided by the number of target binding    sites on that amino acid sequence. The concentration of each amino    acid sequence in the test mix should be high enough to readily    saturate the binding sites for that amino acid sequence on the    target molecules captured on the Biacore chip. The amino acid    sequences in the mixture are at the same molar concentration (on a    binding basis) and that concentration would typically be between    1.00 and 1.5 micromolar (on a binding site basis). Separate    solutions containing A* alone and B* alone are also prepared. A* and    B* in these solutions should be in the same buffer and at the same    concentration as in the test mix. The test mixture is passed over    the target-coated. Biacore chip and the total amount of binding    recorded. The chip is then treated in such a way as to remove the    bound amino acid sequences without damaging the chip-bound target.    Typically this is done by treating the chip with 30 mM HCl for 60    seconds. The solution of A* alone is then passed over the    target-coated surface and the amount of binding recorded. The chip    is again treated to remove all of the bound amino acid sequences    without damaging the chip-bound target. The solution of B* alone is    then passed over the target-coated surface and the amount of binding    recorded. The maximum theoretical binding of the mixture of A* and    B* is next calculated, and is the sum of the binding of each amino    acid sequence when passed over the target surface alone. If the    actual recorded binding of the mixture is less than this theoretical    maximum then the two amino acid sequences are cross-blocking each    other. Thus, in general, a cross-blocking amino acid sequence or    other binding agent according to the invention is one which will    bind to the target in the above Biacore cross-blocking assay such    that during the assay and in the presence of a second amino acid    sequence or other binding agent of the invention the recorded    binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum    theoretical binding, specifically between 75% and 0.1% (e.g. 75% to    4%) of the maximum theoretical binding, and more specifically    between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding    (as just defined above) of the two amino acid sequences or binding    agents in combination. The Biacore assay described above is a    primary assay used to determine if amino acid sequences or other    binding agents cross-block each other according to the invention. On    rare occasions particular amino acid sequences or other binding    agents may not bind to target coupled via amine chemistry to a CM5    Biacore chip (this usually occurs when the relevant binding site on    target is masked or destroyed by the coupling to the chip). In such    cases cross-blocking can be determined using a tagged version of the    target, for example a N-terminal His-tagged version. In this    particular format, an anti-His amino acid sequence would be coupled    to the Biacore chip and then the His-tagged target would be passed    over the surface of the chip and captured by the anti-His amino acid    sequence. The cross blocking analysis would be carried out    essentially as described above, except that after each chip    regeneration cycle, new His-tagged target would be loaded back onto    the anti-His amino acid sequence coated surface. In addition to the    example given using N-terminal His-tagged [target], C-terminal    His-tagged target could alternatively be used. Furthermore, various    other tags and tag binding protein combinations that are known in    the art could be used for such a cross-blocking analysis (e.g. HA    tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies;    biotin tag with streptavidin).-    The following generally describes an ELISA assay for determining    whether an amino acid sequence or other binding agent directed    against a target cross-blocks or is capable of cross-blocking as    defined herein. It will be appreciated that the assay can be used    with any of the amino acid sequences (or other binding agents such    as polypeptides of the invention) described herein. The general    principal of the assay is to have an amino acid sequence or binding    agent that is directed against the target coated onto the wells of    an ELISA plate. An excess amount of a second, potentially    cross-blocking, anti-target amino acid sequence is added in solution    (i.e. not bound to the ELISA plate). A limited amount of the target    is then added to the wells. The coated amino acid sequence and the    amino acid sequence in solution compete for binding of the limited    number of target molecules. The plate is washed to remove excess    target that has not been bound by the coated amino acid sequence and    to also remove the second, solution phase amino acid sequence as    well as any complexes formed between the second, solution phase    amino acid sequence and target. The amount of bound target is then    measured using a reagent that is appropriate to detect the target.    An amino acid sequence in solution that is able to cross-block the    coated amino acid sequence will be able to cause a decrease in the    number of target molecules that the coated amino acid sequence can    bind relative to the number of target molecules that the coated    amino acid sequence can bind in the absence of the second, solution    phase, amino acid sequence. In the instance where the first amino    acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino    acid sequence, it is coated onto the wells of the ELISA plate, after    which the plates are blocked with a suitable blocking solution to    minimize non-specific binding of reagents that are subsequently    added. An excess amount of the second amino acid sequence, i.e.    Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y    target binding sites per well are at least 10 fold higher than the    moles of Ab-X target binding sites that were used, per well, during    the coating of the ELISA plate. Target is then added such that the    moles of target added per well are at least 25-fold lower than the    moles of Ab-X target binding sites that were used for coating each    well. Following a suitable incubation period the ELISA plate is    washed and a reagent for detecting the target is added to measure    the amount of target specifically hound by the coated anti-target    amino acid sequence (in this case Ab-X). The background signal for    the assay is defined as the signal obtained in wells with the coated    amino acid sequence (in this case Ab-X), second solution phase amino    acid sequence (in this case Ab-Y), target] buffer only (i.e. without    target) and target detection reagents. The positive control signal    for the assay is defined as the signal obtained in wells with the    coated amino acid sequence (in this case Ab-X), second solution    phase amino acid sequence buffer only (i.e. without second solution    phase amino acid sequence), target and target detection reagents.    The ELISA assay may be run in such a manner so as to have the    positive control signal be at least 6 times the background signal.    To avoid any artefacts (e.g. significantly different affinities    between Ab-X and Ab-Y for the target) resulting from the choice of    which amino acid sequence to use as the coating amino acid sequence    and which to use as the second (competitor) amino acid sequence, the    cross-blocking assay may to be run in two formats: 1) format 1 is    where Ab-X is the amino acid sequence that is coated onto the ELISA    plate and Ab-Y is the competitor amino acid sequence that is in    solution and 2) format 2 is where Ab-Y is the amino acid sequence    that is coated onto the ELISA plate and Ab-X is the competitor amino    acid sequence that is in solution. Ab-X and Ab-Y are defined as    cross-blocking if, either in format 1 or in format 2, the solution    phase anti-target amino acid sequence is able to cause a reduction    of between 60% and 100%, specifically between 70% and 100%, and more    specifically between 80% and 100%, of the target detection signal    {i.e. the amount of target bound by the coated amino acid sequence)    as compared to the target detection signal obtained in the absence    of the solution phase anti-target amino acid sequence (i.e. the    positive control wells).-   t) An amino acid sequence is said to be “cross-reactive” for two    different antigens or antigenic determinants (such as serum albumin    from two different species of mammal, such as human serum albumin    and cyno serum albumin) if it is specific for (as defined herein)    both these different antigens or antigenic determinants.-   u) By binding that is “essentially independent of the pH” is    generally meant herein that the association constant (K_(A)) of the    amino acid sequence with respect to the serum protein (such as serum    albumin) at the pH value(s) that occur in a cell of an animal or    human body (as further described herein) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the association    constant (K_(A)) of the amino acid sequence with respect to the same    serum protein at the pH value(s) that occur outside said cell.    Alternatively, by binding that is “essentially independent of the    pH” is generally meant herein that the k_(off) rate (measured by    Biacore) of the amino acid sequence with respect to the serum    protein (such as serum albumin) at the pH value(s) that occur in a    cell of an animal or human body (as e.g. further described herein,    e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the k_(off) rate    of the amino acid sequence with respect to the same serum protein at    the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By    “the pH value(s) that occur in a cell of an animal or human body” is    meant the pH value(s) that may occur inside a cell, and in    particular inside a cell that is involved in the recycling of the    serum protein. In particular, by “the pH value(s) that occur in a    cell of an animal or human body” is meant the pH value(s) that may    occur inside a (sub)cellular compartment or vesicle that is involved    in recycling of the serum protein (e.g. as a result of pinocytosis,    endocytosis, transcytosis, exocytosis and phagocytosis or a similar    mechanism of uptake or internalization into said cell), such as an    endosome, lysosome or pinosome.-   v) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   w) The amino acid residues of a Nanobody are numbered according to    the general numbering for V_(H) domains given by Kabat et al.    (“Sequence of proteins of immunological interest”, US Public Health    Services, NIH Bethesda, Md., Publication No. 91), as applied to    V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication); or referred to herein.    According to this numbering, FR1 of a Nanobody comprises the amino    acid residues at positions 1-30, CDR1 of a Nanobody comprises the    amino acid residues at positions 31-35, FR2 of a Nanobody comprises    the amino acids at positions 36-49, CDR2 of a Nanobody comprises the    amino acid residues at positions 50-65, FR3 of a Nanobody comprises    the amino acid residues at positions 66-94, CDR3 of a Nanobody    comprises the amino acid residues at positions 95-102, and FR4 of a    Nanobody comprises the amino acid residues at positions 103-113. [In    this respect, it should be noted that—as is well known in the art    for V_(H) domains and for V_(HH) domains—the total number of amino    acid residues in each of the CDR's may vary and may not correspond    to the total number of amino acid residues indicated by the Kabat    numbering (that is, one or more positions according to the Kabat    numbering may not be occupied in the actual sequence, or the actual    sequence may contain more amino acid residues than the number    allowed for by the Kabat numbering). This means that, generally, the    numbering according to Kabat may or may not correspond to the actual    numbering of the amino acid residues in the actual sequence.    Generally, however, it can be said that, according to the numbering    of Kabat and irrespective of the number of amino acid residues in    the CDR's, position I according to the Kabat numbering corresponds    to the start of FR1 and vice versa, position 36 according to the    Kabat numbering corresponds to the start of FR2 and vice versa,    position 66 according to the Kabat numbering corresponds to the    start of FR3 and vice versa, and position 103 according to the Kabat    numbering corresponds to the start of FR4 and vice versa].-    Alternative methods for numbering the amino acid residues of V_(H)    domains, which methods can also be applied in an analogous manner to    V_(HH) domains from Camelids and to Nanobodies, are the method    described by Chothia et al. (Nature 342, 877-883 (1989)), the    so-called “AbM definition” and the so-called “contact definition”.    However, in the present description, claims and figures, the    numbering according to Kabat as applied to V_(HH) domains by    Riechmann and Muyldermans will be followed, unless indicated    otherwise; and-   x) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74(2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L), domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spatial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   V_(HH) domains and Nanobodies can be expressed from a single        gene and require no post-translational folding or modifications;    -   V_(HH) domains and Nanobodies can easily be engineered into        multivalent and multispecific formats (as further discussed        herein);    -   V_(HH) domains and Nanobodies are highly soluble and do not have        a tendency to aggregate (as with the mouse-derived “dAb's”        described by Ward et al., Nature, Vol. 341, 1989, p. 544);    -   V_(HH) domains and Nanobodies are highly stable to heat, pH,        proteases and other denaturing agents or conditions (see for        example Ewert et al, supra);    -   V_(HH) domains and Nanobodies are easy and relatively cheap to        prepare, even on a scale required for production. For example,        V_(HH) domains, Nanobodies and proteins/polypeptides containing        the same can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibody        fragments;    -   V_(HH) domains and Nanobodies are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) than such conventional 4-chain        antibodies and antigen-binding fragments thereof;

V_(HH) domains and Nanobodies can show so-called cavity-bindingproperties (inter alia due to their extended CDR3 loop, compared toconventional V_(H) domains) and can therefore also access targets andepitopes not accessible to conventional 4-chain antibodies andantigen-binding fragments thereof. For example, it has been shown thatV_(HH) domains and Nanobodies can inhibit enzymes (see for example WO97/49805; Transue et al., Proteins 1998 Sep. 1; 32(4): 515-22; Lauwereyset al., EMBO J. 1998 Jul. 1; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodiesagainst HER2, and in particular Nanobodies against HER2 from awarm-blooded animal, and more in particular Nanobodies against HER2 froma mammal, and especially Nanobodies against human HER2; as well asproteins and/or polypeptides comprising at least one such Nanobody.

In particular, the invention provides Nanobodies against HER2, andproteins and/or polypeptides comprising the same, that have improvedtherapeutic and/or pharmacological properties and/or other advantageousproperties (such as, for example, improved ease of preparation and/orreduced costs of goods), compared to conventional antibodies againstHER2 or fragments thereof, compared to constructs that could be based onsuch conventional antibodies or antibody fragments (such as Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs (see for example the review by Holliger andHudson, Nat. Biotechnol. 2005 September; 23(9):1126-36)), and alsocompared to the so-called “dAb's” or similar (single) domain antibodiesthat may be derived from variable domains of conventional antibodies.These improved and advantageous properties will become clear from thefurther description herein, and for example include, without limitation,one or more of:

-   -   increased affinity and/or avidity for HER2, either in a        monovalent format, in a multivalent format (for example in a        bivalent format) and/or in a multispecific format (for example        one of the multispecific formats described hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased specificity towards HER2, either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow);    -   decreased or where desired increased cross-reactivity with HER2        from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of theinvention, the Nanobodies of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more Nanobodies of the invention and whichmay optionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more amino acid sequences of theinvention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further amino acidsequences that can serve as a binding unit (i.e. against one or moreother antigenic determinants on HER2 and/or against one or more othertargets than HER2), so as to provide a monovalent, multivalent,multiparatopic or multispecific polypeptide of the invention,respectively, all as described herein. In particular, such a protein orpolypeptide may comprise or essentially consist of one or moreNanobodies of the invention and optionally one or more (other)Nanobodies (i.e. directed against one or more other antigenicdeterminants on HER2 and/or against other targets than HER2), alloptionally linked via one or more suitable linkers, so as to provide amonovalent, multivalent, multiparatopic or multispecific Nanobodyconstruct, respectively, as further described herein. Such proteins orpolypeptides may also be in essentially isolated form (as definedherein).

In a Nanobody of the invention, the binding site for binding againstHER2 is preferably formed by the CDR sequences. Optionally, a Nanobodyof the invention may also, and in addition to the at least one bindingsite for binding against HER2, contain one or more further binding sitesfor binding against other antigens, proteins or targets. For methods andpositions for introducing such second binding sites, reference is forexample made to Keck and Huston, Biophysical Journal, 71, October 1996,2002-2011; EP 0 640 130; and WO 06/07260.

As generally described herein for the amino acid sequences of theinvention, when a Nanobody of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human HER2; whereasfor veterinary purposes, it is preferably directed against HER2 from thespecies to be treated.

Also, as with the amino acid sequences of the invention, a Nanobody ofthe invention may or may not be cross-reactive (i.e. directed againstHER2 from two or more species of mammal, such as against human HER2 andHER2 from at least one of the species of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequencesof the invention, the Nanobodies of the invention may generally bedirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of HER2 However, it isgenerally assumed and preferred that the Nanobodies of the invention(and polypeptides comprising the same) are directed against theHerceptin® binding site on HER2 or the Omnitarg binding site on HER2.

As already described herein, the amino acid sequence and structure of aNanobody can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the Nanobodies of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to HER2 with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that:    -   the Nanobodies can bind to HER2 with a k_(on)-rate of between        10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹        and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹        s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   the Nanobodies can bind to HER2 with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of theinvention are such that: a monovalent Nanobody of the invention (or apolypeptide that contains only one Nanobody of the invention) ispreferably such that it will bind to HER2 with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 μM.

The affinity of the Nanobody of the invention against HER2 can bedetermined in a manner known per se, for example using the generaltechniques for measuring K_(D). K_(A), k_(off) or k_(on) mentionedherein, as well as some of the specific assays described herein.

Some preferred IC₅₀ values for binding of the Nanobodies of theinvention (and of polypeptides comprising the same) to HER2 will becomeclear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against HER2, which consists of 4 frameworkregions (FR1 to FR4 respectively) and 3 complementarity determiningregions (CDR1 to CDR3 respectively), in which:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;    and/or

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;    and/or

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) against HER2, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 401-675;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    401-675;    and

CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 951-1225;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    951-1225;    and

CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 1501-1775;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    1501-1775;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when a Nanobody of the invention contains one or more CDR1sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more CDR2sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or 1) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or moreCDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table A-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Nanobodies of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table A-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table A-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable A-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable A-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table A-1, each CDR can be replaced by a CDRchosen from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table A-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table A-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1 Preferred combinations of CDR sequences, preferredcombinations of framework sequences, and preferred combinations offramework and CDR sequences. (“ID” refers to the SEQ ID NO in theattached sequence listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID13D11 126 EVQLVESGGGLV 401 DYGMT 676 WVRRAPGK 951 SINWSGTHTDY 1226HPGGSLRLSCVG GLEWVS ADSVKG SGFSLD 264 127 EVQLVESGGGLV 402 DYAMT 677WVRQAPGK 952 SINWSGTHTDY 1227 QPGGSLRLSCVG GLEWVS ADSVKG SGFSLD 2G2 128EVQLVESGGGLV 403 DYGMT 678 WVRQAPGK 953 SINWSGTHTDY 1228 QPGGSLRLSCVAGLEWVS TDPVKG SGFSLD 13D2 129 EVQLVESGGGLV 404 DYGMT 679 WVRQAPGK 954SINWSGTHTDY 1229 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2D5 130 EVQLVESGGGLV405 DYGMT 680 WVRQAPGK 955 SINWSGTHTDY 1230 QPGGSLRLSCVA GLEWVS ADSVKGSGFSLD 2F4 131 EVQLVESGGGLV 406 DYGMT 681 WVRQAPGK 956 SINWSGTHTDY 1231QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2C3 132 EVQLVESGGGLV 407 DYGMT 682WVRQAPGK 957 SINWSGTHTDY 1232 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17E3 133EVQLVESGGGLV 408 RYTMG 683 WYRQAPGK 958 SIDSSGGTNYA 1233 QAGGSLRLSCVAQRDLVA DSVKG SKMTFM 17H3 134 EVQLMESGGGLV 409 DYGMT 684 WVRQAPGK 959SINWSGTHTDY 1234 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17D2 135 EVQLVESGGGLV410 DYGMT 685 WVRQAPGK 960 SINWSGTHTDY 1235 QPGGSLRLSCVA GLEWVS ADSVKGSGFSLD 2F1 136 EVQLVESGGGLV 411 DYGMT 686 WVRQAPGK 961 SINWSGTHTDY 1236QPGGSLRLSCVA ELEWIS ADSVKG SGFSLD 2E2 137 EVQLVESGGGLV 412 DYGMT 687WVRQAPGK 962 SINWSGTHTDY 1237 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2C2 138EVQLVESGGGLV 413 DYAMT 688 WVRQAPGK 963 SINWSGTHTDY 1238 QPGGSLRLSCVAGLEWVS ADSVKG SGFSLD 2E3 139 EVQLVESGGGLV 414 DYGMT 689 WVRQAPGK 964SINWSGTHTDY 1239 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 13B10 140EVQLVESGGGLV 415 DYGMT 690 WVRQAPGK 965 SINWSGTHTDY 1240 QPGGSLRLSCVAGFEWVS ADSVKG SGFSLD 2D1 141 EVQLVESGGGLV 416 DYGMT 691 WVRQAPGK 966SINWSGTHTDY 1241 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 2H3 142 EVQLVESGGGLV417 DYGMT 692 WVRQAPGK 967 SINWSGTHTDY 1242 QPGGSLRLSCVA GLEWVS ADSVKGSGFSLD 2H1 143 EVQLVESGGGLV 418 DYGMT 693 WVRQAPGK 968 SINWSGTHTDY 1243QPGGSLRLSCVA GLEWVS ADSVRG SGFSLD 2C1 144 EVQLVESGGGLV 419 DYGMT 694WVRQAPGK 969 SINWSGTHTDY 1244 QPGGSLRLSCVA GLEWVS TDSVKG SGFSLD 15C5 145EVQLVESGGGLV 420 DYGMT 695 WVRQAPGK 970 SINWNVTHTDY 1245 QPGGSLKLSCVAGLEWVS AYSVKG SGFSLD 2B3 146 EVQLVESGGGLV 421 DYGMT 696 WVRQAPGK 971SINWSGTHTDC 1246 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 29H2 147 EVQLVESGGGLV422 DYGMT 697 WVRQAPGK 972 SINWSGTHTDY 1247 QPGGSLRLSCVA GLEWVS ADSVKGSGFSLD 17E4 148 EVQLVESGGGLV 423 DYGMT 698 WVRQAPGK 973 SINWSGTHTDY 1248QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 17A2 149 EVQLVESGGGLV 424 DYAMT 699WVRQAPGK 974 SINWSGTHTDY 1249 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 15D1 150EVQLVESGGGLV 425 DYAMT 700 WVRQAPGK 975 SINWSGTHTDY 1250 QPGGSLRLSCVAGLEWVS ADSVKG SGFSLD 17B8 151 EVQLVESGGGLV 426 DYGMT 701 WVRQAPGK 976SINWSGTHTDY 1251 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 15C11 152EVQLVESGGGLV 427 DYGMT 702 WVRQAPGK 977 SINWSGTHTDY 1252 QPGGSLRLSCVAGLEWVS ADSVKG SGFSLD 15G8 153 EVQLVESGGGLV 428 DYGMT 703 WVRQAPGK 978SINWNGTHTDY 1253 QPGGSLKLSCVA GLEWVS AYSVKG SGFSLD 17H14 154EVQLVESGGGLV 429 NYAMT 704 WVRQAPGK 979 SINWSGTHTDY 1254 QPGGSLRLSCVAGLEWVS ADSVKG SGFSLI 27G8 155 EVQLVESGGGLV 430 DYGMT 705 WVRQAPGK 980SINWSGTHTDY 1255 QPGGSLRLSCVA GLEWVS ADSVKG SGFSLD 38C6 156 EVQLVESGGGLV431 DYAMT 706 WVRQAPGK 981 SINWSGTHTDY 1256 QPGGSLRLSCVG GLEWVS ADSVKGSGFSLD 2A4 157 EVQLVESGGGLV 432 DYAMS 707 WVRQAPGK 982 AINWSGSHRN 1257QPGGSLRLSCAA GLEWVS YADSVKG SGFIFD 15G7 158 EVQLVESGGGLV 433 DYAMS 708WVRQAPGK 983 AINWSGTHRN 1258 QPGGSLRLSCAA GLEWVS YADSVKG SGFIFD 15B7 159EVQLVESGGGLV 434 DYAMS 709 WVRQAPGK 984 AINWSGSHRN 1259 QPGGSLKLSCAAGLEWVS YADSVKG SGFIFD 5G4 160 EVQLVESGGGLV 435 DYAMS 710 WVRQAPGK 985SINWSGSHRN 1260 QPGGSLTLSCAG GLEWVS YADSVKG SGFIFD 13B2 161 EVQLVESGGSLV436 DYAMS 711 WVRQAPGK 986 SINWSGTHKDY 1261 QPGGSLRLSCAA GLEWIS ADSVKGSGFTFD 2E5 162 EVQLVESGGSLV 437 DYAMS 712 WVRQAPGK 987 SINWSGTHTDY 1262QPGESLRLSCAA GLEWIS ADSVKG SGFTFD 15G1 163 EVQLVESGGSLV 438 DYAMS 713WVRQAPGK 988 SINWSGTHTDY 1263 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 27B1 164EVQLVESGGSLV 439 DYAMS 714 WVRQAPGK 989 SINWSGTHTDY 1264 QPGGSLRLSCAAGLEWIS ADSVKG SGFTFD 17E7 165 EVQLVESGGSLV 440 DYAMS 715 WVRQVPGK 990SINWSGTHTDY 1265 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 17D8 166 EVQLVESGGSLV441 DYAMS 716 WVRQAPGK 991 SINWSGTHTDY 1266 PPGGSLRLSCAV GLEWVS TDSVKGSGFTFD 5F8 167 EVQLVESGGSLV 442 DYALS 717 WVRQAPGK 992 SINWSGTHTDY 1267QPGGSLRLSCAA GLEWIS ADSVKG SGFTFD 2D4 168 EVQLVESGGSLV 443 DYAMT 718WVRQAPGK 993 SINWSGTHTDY 1268 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 13D8 169EVQLVESGGSLV 444 DYAMT 719 WVRQASGK 994 SINWSGTHTDY 1269 QPGGSLRLSCAAGLEWVS TDSVKG SGFTFD 17G8 170 EVQLVESGGSLV 445 DYAMS 720 WVRQAPGK 995SINWSGTHTGY 1270 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2H4 171 EVQLVESGGSLV446 DYAMT 721 WVRQAPGK 996 SINWSGTHTDY 1271 QPGGSLRLSCAA GLEWVS TDSVKGSGFTFD 2F3 172 EVQLVESGGSLV 447 DYAMT 722 WVRQAPGK 997 SINWSGTHTDY 1272QPGGSLRLSCAA GLEWVS TGSVKG SGFTFD 2F5 173 EVQLVESGGSLV 448 DYAMS 723WVRQAPGK 998 SINWSGTHTDY 1273 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 30E10174 KVQLVESGGSLV 449 DYAMT 724 WVRQAPGK 999 SINWSGTHTDY 1274PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 29H1 175 EVQLVESGGSLV 450 DYAMS 725WVRQAPGK 1000 SINWSGTHTGY 1275 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 17E2176 EVQLVESGGSLV 451 DYGMS 726 WVRQAPGK 1001 SINWSGTHTDY 1276PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2B1 177 EVQLVESGGSLV 452 DYAMT 727WVRQAPGK 1002 SINWSGTHTDY 1277 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2A5 178EVQLVESGGGLV 453 DYAMT 728 WVRQAPGK 1003 SINWSGTHTDY 1278 QPGGSLRLSCATGLEWVS TDSVKG SGFTFD 13C12 179 EVQLVESGGSLV 454 DYAMT 729 WVRQAPGK 1004SINWSGTHTDY 1279 QPGGSLRLSCAT GLEWVS TDSVKG SGFTFD 17E10 180EVQLVESGGSLV 455 DYAMT 730 WVRQAPGK 1005 SINWSGTHTDC 1280 QPGGSLRLSCAAGLEWVS TDSVKG SGFTFD 27D4 181 EVQLVESGGSLV 456 DYAMT 731 WVRQASGK 1006SINWSGTHTDY 1281 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 15F9 182 EVQLVESGGSLV457 DYAMT 732 WVRQAPGK 1007 SINWSGTHTDY 1282 QPGGSLRLSCAA GLEWVS TGSVKGSGFTFD 30H9 183 EVQLVESGGSLV 458 DYAMT 733 WVRQAPGK 1008 SINWSGTHTDY1283 QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 39C1 184 EVQLVESGGSLV 459 DYGMS734 WVRQAPGK 1009 SINWSGTHTDY 1284 PPGGSLRLSCAA GLEWVS TDSVKG SGFTFD27G2 185 EVQLVESGGSLV 460 DYAMT 735 WVRQTPGK 1010 SINWSGTHTDY 1285QPGGSLRLSCAA GLEWVS TDSVKG SGFTFD 2D3 186 EVQLVESGGSLV 461 DYAMS 736WVRQVPGK 1011 SINWSGTHTDY 1286 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD 5F7 187EVQLVESGGGLV 462 INTMG 737 WYRQAPGK 1012 LISSIGDTYYAD 1287 QAGGSLRLSCAAQRELVA SVKG SGITFS 118N121_A1_4_OK/ 188 EVQLVESGGGFV 463 EYAAA 738WFRQSPGK 1013 GIMWDGRSLF 1288 1-127 QTGGSPRLSCAA ERDLVA YADSVKG SGRSFS47D5 189 KVQLVESGGGLV 464 FNDMA 739 WYRQAPGK 1014 LISRVGVTSSA 1289QPGGSLRLSCAA QRELVA DSVKG SGSIFG 14B11 190 EVQLVESGGGLV 465 SYGMG 740WFRQVPGK 1015 TINWSGVTAYA 1290 QAGGSLRLSCAA EREFVA DSVKG SGSTFS 14B10191 EVQLVESGGGLV 466 SYGMG 741 WFRQAPGK 1016 TINWSGVTAYA 1291QAGGSLRLSCAV EREFVA DSIKG NSRTFS 14B4 192 EVQLVESGGGLV 467 SYGMG 742WFRQAPGK 1017 TINWSGVTAYA 1292 QAGGSLRLSCAV DREFVA DSIKG SSRAFS 14C11193 EVQLVESGGGLV 468 SYGMG 743 WFRQAPGK 1018 TINWSGATAYA 1293QAGGSLRLSCAV EREFVA DSIKG NSRTFS 14B5 194 EVQLVESGGGLV 469 SYGMG 744WFRQAPGK 1019 TINWSGVTAYA 1294 QAGGSLRLSCAV DREFVA DSIKG SSRAFS 14C6 195EVQLVESGGGSV 470 SYGMG 745 WFRQAPGK 1020 TINWSGVTAYA 1295 QAGGSLRLSCVAERAFVA DSVKG SEGTFS 14A4 196 EVQLVESGGGSV 471 SYGMG 746 WFRQAPGK 1021TINWSGVNAYA 1296 QAGSSLTLSCVAS ERAFVA DSVKG EGTFS 14B3 197 EVQLVESGGGLV472 SYGMG 747 WFRQAPGK 1022 TINWSGVNAYA 1297 QPGGSLTLSCVA ERAFVA DSVKGSEGTFS 14C1 198 EVQLVESGGGSV 473 SYGMG 748 WFRQAPGK 1023 TINWSGVTAYA1298 QAGGSLRLSCAA ERAFVA DSVKG SGSTFS 14A12 199 EVQLVKSGGGLV 474 SYGMG749 WFRQAPGK 1024 TINWSGVTAYA 1299 QAGGSLRLSCAA EREFVA DSVKG SERTFS 14A2200 EVQLVESGGGLV 475 SYGMG 750 WFRQAPGK 1025 TINWSGVTAYA 1300QAGGSLRLSCAA EREFVA DSVKG SERTFS 14A1 201 EVQLVESGGGSV 476 SYGMG 751WFRQAPGK 1026 TINWSGVTAYA 1301 QAGGSLRLSCAA EREFVA DSVKG SERTFS 17C3 202EVQLVESGGGLV 477 RYDMG 752 WYRQAPGQ 1027 AISGAGDINYA 1302 QAGGSLRLSCAAQREWVA DSVKG NGLTFR 46D3 203 KVQLVESGGGLV 478 EYSMG 753 WFRQAPGK 1028TISWNYGYTYY 1303 QAGGSLRLSCAA EREFVA SDSVKG SGRTFT 27H5 204 EVQLVESGGGLV479 DYGIG 754 WFRQASGK 1029 CITSSDGSTYY 1304 QAGGSLRLSCAA EREGVS ADSVKGSGFTFD 17C2 205 EVQLVESGGGLV 480 SYAMS 755 WVRQAPGK 1030 AVDSGGGRTD 1305QPGGSLRLSCAA GLEWVS YAHSVKG SGFAFS 17D11 206 EVQLVESGGGLV 481 TSAMG 756WFRQAPGK 1031 TISRGGSATYY 1306 QAGGSLRLSCTA EREFVA ADSLKG SGRTSS 15A6207 EVQLVESGGGLV 482 TRTMA 757 WYRQAPGK 1032 TISSHGLPVYA 1307QAGGSLRLSCVT QRDWVA DSVKG SRRPAS 17B6 208 EVQLVESGGGLV 483 TRTMA 758WYRQAPGK 1033 TIGTSGPPRYA 1308 QPGGSLRLSCAA QRDWVA DSVKG SRIPFS 17C5 209EVQLVESGGGLV 484 TRTMA 759 WYRQAPGK 1034 TISSHGLPVYA 1309 QAGGSLRLSCVTQRDWVA DSVKG SRRPAS 15E11 210 EVQLVESGGGLV 485 SRTMA 760 WYRQAPGK 1035TISARGMPAYE 1310 QAGGSLRLSCVA QRDWVA DSVKG SRIPFS 15C2 211 EVQLVESGGGLV486 TRTMA 761 WYRQAQGK 1036 TISSHGLPVYA 1311 QAGGSLRLSCVT QRDWVA DSVKGSRRPAS 2A3 212 EVQLVESGGGLV 487 TRTMA 762 WYRQAPGK 1037 TIRNGAPVYAD 1312QAGGSLNLSCVA PRDWVA SVKG SGIPFS 27A5 213 EVQLVESGGGLV 488 TRTMA 763WYRQPPGN 1038 TIRSGAPVYAD 1313 QAGGSLNLSCVA ERDWVA SVKG SGIPFS 2C5 214EVQLVESGGGLV 489 TRTMA 764 WYRQTPGK 1039 TIRSGTPVYAD 1314 QAGGSLNLSCVASRDWVA SVKG SGIPFS 27G5 215 EVQLVESGGGLV 490 IRTMA 765 WYRQTPGN 1040TIGSSGTPAYA 1315 QPGGSLRLSCVA QRDWLA DSVKG SRIPAS 13A9 216 EVQLVESGGGLV491 IRTMA 766 WYRQAPGK 1041 TIGTGGTPAYA 1316 QAGGSLRLSCVA QRDWVA DSFKGSRIPAS 29E9 217 EVQLVESGGGLV 492 IRTMA 767 WYRQTPGN 1042 TIGSSGTPAYA1317 QPGGSLRLSCVA QRDWLA DSVKG SRIPAS 15D8 218 EVQLVESGGGLV 493 IRTMA768 WYRQTPGN 1043 TIGSSGTPAYA 1318 QPGGSLKLSCVA QRDWLA DSVKG STIPAS 15G4219 EVQLVESGGGLV 494 SRTMA 769 WYRQAPGK 1044 TIGTHGTPLYA 1319QAGGSLRLSCVA TRDWVA DSVKG SGIPFR 15D12 220 EVQLVESGGGLV 495 RYVMG 770WYRQGPGK 1045 TVNDGGTTSY 1320 QAGESLRLSCATS QRELVA ADSVKG GITFK 15E12221 EVQLMESGGGLV 496 RYDMG 771 WYRQAPGQ 1046 AISGAGDINYA 1321QAGGSLRLSCAA QREWVA DSVKG NGLTFR 13D7 222 EVQLVESGGGLV 497 RYDMG 772WYRQAPGQ 1047 AISGAGDINYA 1322 QAGGSLRLSCAA QREWVA DSVKG NGLTFR 13A8 223EVQLVESGGGLV 498 FSRRTMA 773 WYRQAPGK 1048 TIAGDGSTVYA 1323 QPGGSLRLSCAAQRDWVA DSMKG SGLGIA 15A4 224 EVQLVESGGGLV 499 FSRRTMA 774 WYRQAPGK 1049TIAGDGSTVYA 1324 QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 17F7 225 EVQLVESGGGLV500 IRVMA 775 WYRQPPGK 1050 TISSDGTANYA 1325 QAGGSLRLSCVA QRDWVG DSVKGSGIAQS 15C8 226 EVQLVESGGGLV 501 IRTMA 776 WYRQAPGK 1051 TSDSGGTTLYA1326 QAGGSLRLSCAA QRDWVA DSVKG SGIAFR 17A10 227 EVQLVESGGGLV 502 RAIA777 WYRQAPGK 1052 TSGTGYGATY 1327 QAGGSLRLSCVA QRDWVA DDSVKG SGIPSI 27D3228 EVQLMESGGGLV 503 FSRRTMA 778 WYRQAPGK 1053 TIAGDGSTVYA 1328QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 13B12 229 EVQLVESGGGLV 504 IRTMA 779WYRQAPGK 1054 TIGSDGTTJYAD 1329 QAGGSLRLSCAA QRDWVA SVKG SGIAFR 15B2 230EVQLVESGGGLV 505 IRAMA 780 WYRQAPGR 1055 TIYSPSGSAVY 1330 QAGGSLRLSCVVQRDWVA ADSVKG SGIPSS 15B11 231 EVQLVESGGGSV 506 IRAMA 781 WYRQAPGR 1056TIYSRSGGAVY 1331 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 13C9 232 EVQLVESGGGLV507 HAMA 782 WYRQAPGK 1057 TTYSRGGTTYN 1332 QAGGSLRLSCVA QRDWGA DSAKGSGIPSI 17D5 233 EVQLVESGGGLV 508 IRTMA 783 WYRQAPGK 1058 SIGTRGAPVYA1333 QPGGSLRLSCAA QRDWVA DSVNG SGIIGT 27B5 234 EVQLVESGGGLV 509 IRTMA784 WYRQAPGK 1059 TSDSGGTTLYA 1334 QAGGSLRLPCAA QRDWVA DSVKG SGIAFR 27C7235 EVQLVESGGGLV 510 IRTMA 785 WYRQAPGK 1060 TSDSGGTTLYA 1335QAGGSLRLSCAA QRDWVA DSVKG SGIAFR 13D4 236 EVQLVESGGGLV 511 IRAMA 786WYRQAPGR 1061 TIYSPSGSAVY 1336 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 15G5237 EVQLVESGGGLV 512 IRAMA 787 WYRQAPGR 1062 TIYSPSGSAVY 1337QAGGSLRLSCVV QRDWVA ADSVKG SGIPST 13C4 238 EVQLVESGGGLV 513 IRAMA 788WYRQAPGR 1063 TIYSPSGSAVY 1338 QAGGSLRLSCVV QRDWVA ADSVKG SGIPSS 46G1239 EVQLVESGGGLV 514 DDAMG 789 WFRQAPGK 1064 SLYLNGDYPYY 1339QAGGSLRLSCAA ERECVA ADSVKG SGRTFS 46E4 240 EVQLVESGGGLV 515 DDAVG 790WFRQAPGK 1065 SMYLDGDYPY 1340 QAGGSLRLSCAA ERECVA YADSVKG SGRAFK 17B5241 EVQLVESGGGLV 516 TDMMG 791 WYRQAPGK 1066 SITKFGSTNYA 1341QTGGSLRLSCAA QREFVA DSVKG SGSTFR 15C9 242 EVQLVESGGGLV 517 LRAMA 792WYRQAPGR 1067 TSSNTGGTTYD 1342 QAGGSLKLSCVN QRDWVA DSVKG SGIPST 13D10243 EVQLVESGGGLV 518 DSNAIG 793 WFRQAPGK 1068 CIASSDGSTYY 1343QPGGSLRLSCAA EREEVS AESVKG SSVITL 17C6 244 EVQLVESGGGLV 519 LDIMA 794WYRQAPEK 1069 SVSGGGNSDY 1344 QAGGSLTLSCAA QRELVA ASSVKG SGSTSS 15A2 245EVQLVESGGGLA 520 TRVMA 795 WYRQTPGK 1070 SMRGSGSTNY 1345 QAGGSLSLSCAAQREFVA ADSARG SGRFFS 17A8 246 EVQLVESGGGLV 521 TRVMA 796 WYRQTPGK 1071SMRGSGSTNY 1346 QAGGSLSLSCAA QREFVA ADSVRG SGRFFS 15G10 247 EVQLVESGGGLV522 TRVMA 797 WYRQTPGK 1072 SMRGSGSTNY 1347 QAGGSLSLSCAA QREFVA ADSARGSGRFFS 27A3 248 EVQLVESGGGLV 523 TRVMA 796 WYRQTPGK 1073 SMRGSGSTNY 1348QAGGSLSLSCVA QREFVA ADSVRG SGRFFS 17H10 249 EVQLVESGGGLV 524 TRVMA 799WYRQTPGN 1074 TIHSSGSTIYAD 1349 QAGGSLSLSCSA QREFVA SVRG SGRFFS 30D10250 EVQLVESGGGLV 525 IRTMA 800 WYRQPPGN 1075 TIGSNGFATYP 1350QAGGSLTLSCTAS QREWVA DSVKG ETTVR 15H4 251 EVQLVESGGGLV 526 FNTVA 801WYRQAPGE 1076 TISRQGMSTYP 1351 QAGGSLTLSCAP QREWVA DSVKG SESTVS 17B7 252EVQLVESGGGLV 527 FRTMA 802 WYRQAPGK 1077 TIGSDGLANYA 1352 QAGGSLRLSCAAQREWVA DSVKG SGIISS 15D2 253 EVQLVESGGGLV 528 IRAMA 803 WYRQAPGK 1078TIGSSGHPVYT 1353 QAGGSLRLSCVV QRDWVA DSVKG SGVFGP 17G5 254 EVQLVESGGGLV529 FSSRTMA 804 WYRQAPGK 1079 TIGSGGTTNYA 1354 QPGGSLRLSCAA QRDWVA DSVKGSGIGIA 15B6 255 EVQLVESGGGLV 530 FRTMA 805 WYRQAPGN 1080 TIGSAGLASYA1355 QPGGSLRLSCAA QRDWVA DSVRG SGIIGS 27F2 256 EVQLVESGGGLV 531 FRTLA806 WYRQAPGK 1081 TISSAGGTAYA 1356 QAGGSLRLSCAA QRDWVA DAVKG SGIISS 17F5257 EVQLVESGGGLV 532 FSRRTMA 807 WYRQAPGK 1082 TIAGDGSTVYA 1357QPGGSLRLSCAA QRDWVA DSMKG SGLGIA 17B2 258 EVQLVESGGGLV 533 NYAMT 808WVRQAPGK 1083 GVGGDGVGSY 1358 QPGGSLRLSCAG GLEWVS ADSVKG SGFTFS 27H4 259EVQLVESGGGLV 534 RYTMG 809 WYRQAPGK 1084 SIDASGGTNYA 1359 QAGGSLRLSCVAQRDLVA DSVKG SKMTFM 13A4 260 EVQLVESGGGLV 535 RYTMG 810 WYRQAPGK 1085SIDSSGGTNYA 1360 QAGGSLRLSCVA QRDLVA DSVKG SKMTFM 2A1 261 EVQLVESGGGLV536 RYIMD 811 WYRQAPGK 1086 SINSDGSTGYT 1361 QAGGSLRLSCVA QRELVA DSVKGSKITFR 15E10 262 EVQLVESGGGLV 537 RYTMG 812 WYRQAPGK 1087 EISSADEPSFA1362 QAGGSLKLSCVA ERELVA DAVKG SGITFF 27E7 263 EVQLVESGGGLV 538 RYDMG813 WYRQFPGK 1088 TILSEGDTNYV 1363 QAGGSLRLSCAA ERELVA DPVKG SGITFR 47E5264 EVQLVESGGGLV 539 FDSMG 814 WYRQAPGN 1089 IISNGGTTSYR 1364QAGGSLRLSCAA ERILVA DSVKG SASIFG 2G4 265 EVQLVESGGGLV 540 HNAMG 815WYRQAPGK 1090 YITINGIANYVD 1365 QAGGSLRLSCAA QRELVT SVKG SGNIFS 14D4 266EVQLVESGGGLV 541 TYVMG 816 WFRQAPGD 1091 HIFRSGITSYAS 1366 QAGDSLRLSCAAGREFVA SVKG SGRALD 17A5 267 EVQLVESGGGLV 542 DYSMS 817 WVRQATGK 1092GISWNGGSTN 1367 QPGGSLRLSCAA GLEWVS YADSVKG SGFTFD 15D10 268EVQLVESGGGLV 543 SYRMY 818 WVRQAPGK 1093 AIKPDGSITYYA 1368 QPGGSLKLSCAAGLEWVS DSVKG SGFTFS 13C2 269 EVQLVESGGGLV 544 INRMA 819 WYRQSPGK 1094AVDNDDNTEY 1369 QAGGSLRLSCAA QRELVA SDSVAG SGSTFS 17G11 270 EVQLVESGGGLV545 INRWG 820 WYRQAPGK 1095 AIDDGGNTEYS 1370 QAGGSLRLSCAA QRELVA DFVNGSGSTFS 17A3 271 EVQLVESGGGLV 546 FDNN 821 WYRQAPGK 1096 TIAHDGSTNYA 1371QAGGSLSLSCAA QRELVA NSVKG SATLHR 27B7 272 EVQLVESGGGLV 547 SYAMS 822WVRQAPGK 1097 AISSGGGSITTY 1372 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 17A6273 EVQLVESGGGLV 548 SYAMS 823 WVRQAPGK 1098 AISSGGGSITTY 1373QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 17D7 274 EVQLVESGGGLV 549 YCAIG 824WFRQAPGK 1099 CISSSDGSTYY 1374 QPGGSLRLSCAA EREGVS ADSVKG SGFTLD 46D4275 EVQLVESGGGLV 550 DYAMS 825 WVRQAPGK 1100 SINWSGTHTDY 1375QPGGSLRLSCAA GLEWVS AEDMKG SGFIFD 27B3 276 EVQLVESGGGLV 551 IRTMA 826WYRQPPGN 1101 TIGSNGFATYP 1376 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 27E5 277EVQLVESGGGLV 552 IRTMA 827 WYRQPPGN 1102 TIGSNGFATYP 1377 QAGGSLTLSCTASQREWVA DSVKG ETTVR 27D6 278 EVQLVESGGGLV 553 IRTMA 828 WYRQPPGN 1103TIGSNGFATYP 1378 QAGGSLTLSCTAS QREWVA DSVKG ETTVR 30D10 279 EVQLVESGGGLV554 IRTMA 829 WYRQPPGN 1104 TIGSNGFATYP 1379 QAGGSLTLSCTAS QREWVA DSVKGETTVR 47G11 280 EVQLVESGGGLV 555 PMG 830 WFRQAPGK 1105 AIGSGDIITYYA 1380QPGGSLRLSCAA EREFVA DSVKG SGRIFY 27C3 281 EVQLVESGGGLV 556 DYATS 831WVRQAPGK 1106 AINSGGGSTYY 1381 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFD11A101/1- 282 EVQLVESGGGLV 557 AMG 832 WFRQAPGK 1107 AISRSPGVTYY 1382120 QAGGSLRLSCAA EREFVA ADSVKG SGRTFN 11A22/1- 283 EVQLVESGGGLV 558SYAMA 833 WFRQAPGT 1108 GIRWSDGSTY 1383 122 QAGGSLRLSCAA EREFIA YADSVKGSGRTFS 12D44/1- 284 KVQLVESGGGLV 559 SYAMA 834 WFRQAPGT 1109 GIRWSDGSTY1384 122 QAGGSLRLSCAA EREFIA YADSVKG SGRTFS 12E11/1- 285 EVQLVESGGGLV560 SYAMA 835 WFRQAPGK 1110 GIRWSDGSTY 1385 122 QAGGSLRLSCAA EREFVGYADSVKG SGRTFS 13G111/ 286 EVQLVESGGGLV 561 SYAMG 836 WFRQAPGK 1111AIRWSGGNTY 1386 1-123 QAGGSLRLSCAA ERAFVA YADSVKG SGRTFS 13F71/1- 287EVQLVESGGGLV 562 NYALA 837 WFRQAPGK 1112 AINWRSGGST 1387 123QAGGSLRLSCVA EREFVA YYADSVKG SGRTFS 14H61/1- 288 EVQLVESGGGLV 563 RFAMG838 WFRQAPGK 1113 AVRWSDDYTY 1388 122 QAGGSLRLSCAA EREFVA YADSVKG SGRTFS22B12/1- 289 EVQLVESGGGLV 564 SYAMA 839 WFRQAPGK 1114 GINKSGGITHS 1389124 QAGGSLRLSCAA EREFVA ADSVKG SGRTFS 14H71/1- 290 EVQLVESGGGLV 565SLTMA 840 WFRQAPGK 1115 NIKWSGDRIVY 1390 123 QAGGSLRLSCEA EREFVA ADSVKGSGLTIS 12D51/1- 291 EVQLVESGGGLV 566 IKSMG 841 WYFRQAPGK 1116VIISSGTTTYAD 1391 120 QPGGSLRLSCAA QRELAA SVKG SGSAFS 11A111/1- 292EVQLVESGGGLV 567 SSLMG 842 WFRQAPGK 1117 AITDNGGSTYY 1392 126QAGGSLGLSCAA EREFVA ADSVKG AGRTFS 13G71/1- 293 EVQLVESGGGLV 568 SYAMG843 WFRQAPGK 1118 AITSSGSTNYA 1393 124 QAGGSLRLSCAA ERDFVA DSVKG SGRAFS13G74/1- 294 EVQLVESGGGLV 569 TYASMG 844 WFRQTPGK 1119 AITSSGSTNYA 1394125 QAGGSLRLSCAT EREFVA DSVKG SGRTFS 11A71A/ 295 EVQLVESGGGLV 570 IITMG845 WYRQRPGK 1120 TINSGGDTNYA 1395 1-116 QPGGSLRLSCAA PREWVG GSVKGSGNIDG 22B101/1- 296 EVQLVESGGGLV 571 DYAIG 846 WFRQAPGK 1121AISSSGISTIYG 1396 123 QTGGSLRLSCAA EREFVA DSVKG SGPTFS 11B42/1- 297EVQLVESGGGLV 572 NHIMG 847 WFRQAPGK 1122 HITWNGGSTYY 1397 123QAGDSLRLSCAA ERELIA ADSVKG SGFTFS 13E111/1- 298 EVQLVESGGGLV 573 DYAIG848 WFRQAPGK 1123 AISGWSGGTT 1398 124 QAGSSLRLSCALS EREFVA NYADSVKGGRTFS 14H12/1- 299 EVQLVESGGGLV 574 SAGVG 849 WFRQAPGK 1124 AISWNGVTIYY1399 125 QAGGSLRLSCIAS ERDFVA ADSVKG ERTFS 13G101/ 300 EVQLVESGGGLV 575RSRVA 850 WFRQAPGK 1125 VISGVGTSYAD 1400 1-123 QPGDSLRLSCSA EREFVT SVKGSEGTLS 13G41/1- 301 EVQLVESGGGLV 576 ADVMG 851 WYRQAPGK 1126SISSGSAINYAD 1401 121 QPGGSLTLSCVG QREFVA SVKG SGRRFS 22B910/1- 302EVQLVESGGGLV 577 MNDMG 852 WYRQAPGK 1127 TLTSAGNTNYA 1402 121QPGGSLPLSCAA ORERVA DSVKG SGSIFR 21A81/1- 303 EVQLVESGGGLA 578 GMA 853WFRRAPGK 1128 GIAWNGASIGS 1403 122 QAGGSLRLSCAV EREFVA ADSVRG FGRSRY21A92/1- 304 EVQLVESGGGQV 579 TRAMG 854 WFRQAPEK 1129 GITMSGFNTRY 1404127 QAGGSLRLSCTE EREFVA ADSVKG SGRAFN 22C712/1- 305 EVQLVESGGGLV 580NYAMG 855 WFRQAPGK 1130 GISWSGGHTF 1405 123 QAGGSLGLSCAA EREFVA YADSVKGSGRTFS 11A13/1- 306 EVQLVESGGGLV 581 SYAMG 856 WFRQAPGK 1131 TIDWSGDTAFY1406 125 QAGDSLRLSCVA EREFVA ADSVKG SGGTFG 13G93/1- 307 EVQLVESGGGLA 582AYAMG 857 WFRQAPGK 1132 AVSWDGRNTY 1407 123 QAGDSLRLSCVD EREFVA YADSVKGSGSSFS 12C52/1- 308 EVQLVESGGGLV 583 SDTMA 858 WFRQAPGK 1133 RVSWIRTTYYS1408 118 QAGGSLRLSCAV EREFVA DSVKG SGGTFE 12C61/1- 309 EVQLVESGGGLV 584SNAMA 859 WFRQAPGN 1134 AIGWSGASTYY 1409 126 QAGGSLRLSCAA ERELVS IDSVEGSGRTFS 21A61/1- 310 EVQLVESGGGLV 585 TYTMG 860 WFRQAPGK 1135 AIRWSGGTTFY1410 125 QAGDSLRLSCVA EREFVA GDSVKG SGDSFN 11A121/1- 311 EVQLVESGGGLV586 SYSMG 861 WFRQAPGK 1136 AITWNGTRTYY 1411 126 QAGGSLRLSCVV DREFVSRDSVKG SEGTFS 11A91/1- 312 EAQLVESGGGLV 587 TTMG 862 WFRQAPGK 1137AIRWSGGSAFY 1412 124 QAGGSLRLSCTA EREFVA ADSVKG SGRTYS 13G72/1- 313EVQLVESGGGLV 588 SYAMG 863 WFRQAPGK 1138 AITSSGSTNYA 1413 118QAGGSLRLSCAA ERDFVA DSVKG SGRAFS 13E81/1- 314 EVQLVESGGGLV 589 VYHMA 864WFRQAPGK 1139 AIRSSGGLFYA 1414 124 QAGGSLRLSCAA EREFVA LSVSG SGGTFS11B31/1- 315 EVQLVESGGGLV 590 VYHMG 865 WFRQAPGK 1140 AIRSGGTTLYE 1415124 QAGGSLRLSCAA EREFVA DSVKG SGGAFG 13G81/1- 316 EVQLVESGGGLV 591 VYHMG866 WFRQAPGK 1141 VIRSGGTTLYA 1416 124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG21A53/1- 317 EVQLVESGGGLV 592 VYHMG 867 WFRQAPGK 1142 AIRSGGTTLYE 1417124 QAGGSLELSCAA EREFVA DSVKG SGGAFG 14H51/1- 318 EVQLVESGGGLV 593 VYTMA868 WFRQAPGK 1143 AIRSGATTLYE 1418 124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG21A21/1- 319 EVQLVESGGGLV 594 VYHMG 869 WFRQAPGT 1144 VIRSGGTTLYE 1419124 QAGGSLRLSCAA EREFVA DSVKG SGGTFG 21A111/1- 320 EVQLVESGGGLV 595PYTMA 870 WFRQAPGK 1145 VTRSGGTTFYA 1420 124 QAGGSLKLSCAV EREFVA DSAKGSGRTIV 22B1212/ 321 EVQLVESGGGLV 596 SYAMS 871 WVRQAPGK 1146 AINSGGGSTSY1421 1-122 QPGGSLRLSCAA GLEWVS ADSVKG SGFTFS 11A31/1- 322 EVQLVESGGGLV597 SGVMA 872 WFRQSPGE 1147 LITRNGETKKT 1422 120 QAGGSLRLSCAA EREFLAADSVKG SGGTFS 13E51/1- 323 EVQLVESGGGLV 598 GYAMG 873 WFRQAPGK 1148AIRWSGGITYY 1423 128 QAGGSLRLSCAA EREFVA ADSVKG SRHTFS 12D121/1- 324EVQLVESGGGLV 599 TYGMG 874 WFRQAPGK 1149 AISRSGTGTYY 1424 126QTGGSLRLSCAA AREFVA AGSMKG SGRAFS 13F121/1- 325 EVQLVESGGGLV 600 DYTMG875 WFRQTPGK 1150 RVWWNGGSA 1425 119 QAGGSLRLSCAA EREFVA YYADSVKG SGRSFN13G121/ 326 EVQLVESGGGLV 601 SAAMG 876 WFRQAPGK 1151 AISPIGSSKYYA 14261-127 RAGTSLRLSCADS EREFVS DSVKG ARTFS 22B41/1- 327 EVQLVESGGGLV 602GDVIG 877 WFRQAPGK 1152 AISTSGGGTDS 1427 124 QPGGSLRLSCTV EREFVA ADSVKGFGRTFS 12D71/1- 328 EVQLVESGGGLV 603 TMG 878 WFRQAPGK 1153 AITWSGDSTNF1428 125 QAGGSLGLSCAA EREFVT ADSVKG SGRTVS 13F42/1- 329 EVQLVESGGGLV 604TTGVG 879 WFRQAPGK 1154 TIFVGGTTYYS 1429 111 QAGGSLRLSCVA GRESVA DSVKGSGRTLS 12C101/1- 330 EVQLVESGGGLV 605 TTGVG 880 WFRQAPGK 1155TIFVGGTTYYS 1430 111 QAGGSLRLSCVA ERESVA DSVKG SGRTLS 14H91/1- 331EVQLVESGGGLV 606 RDVMG 881 WFRQAPGK 1156 AKTWSGASTY 1431 127QAGGSLRLSCAA EREFVA YADSVRG SGRTFS 13F41/1- 332 EVQLVESGGGLV 607 TTGVG882 WFRQAPGK 1157 TIFVGGTTYYS 1432 111 QAGGSLRLSCVA ERESVA DSVKG SGRTLS14H21/1- 333 EVQLVESGGGLV 608 SYHIG 883 WFRQAPGN 1158 AITWNGASTSY 1433125 QAGGSLRLSCVR EREFVA ADSVKG SGGYFG 22B610/1- 334 EVQLVESGGGLV 609INAMG 884 WYRPAPGK 1159 RITSTGSTNYA 1434 120 QPGGSLRLSCAA QRELVA DSVKGSGSIFS 12C32/1- 335 EMQLVESGGGLV 610 TYTMA 885 WFRQAPGK 1160 AIKSSDNSTSY1435 127 QAGGSLRLSCAT EREFVV RDSVKG SERTFS 12D61/1- 336 EVQLVESGGGLV 611PNVVG 886 WYRQAPGK 1161 AVTSGGITNYA 1436 116 QPGGSLRLSCAA QRELVA DSVKGSRSIFS 13G31/1- 337 EVQLVESGGGLV 612 RYKMG 887 WFRQAPGK 1162 ASRWSGGIKY1437 125 QAGGSLRLSCAA EREFVA HADSVKG SGGTFS 22C65/1- 338 EVQLVESGGGLV613 SYAMG 886 WFRQAPGK 1163 AIRWSGSATDY 1438 124 QAGGSLRLSCAV EREFVASDSVKG SGFLFD 11A71/1- 339 EVQLVESGGGLV 614 VSVMG 889 WYRLAPGN 1164TITADGITNYAD 1439 125 QPGGSLRLSCAA QRELVA SVKG SRSIRS 11B91/1- 340EVQLVESGGALV 615 INTMG 890 WYRQAPGN 1165 AVTEGGTTSYA 1440 125QPGGSLRLSCAA QREFVA ASVKG SGSIRS 11A81/1- 341 EVQLVESGGALV 616 INIMG 891WYRQAPGK 1166 AVTEDGSINYA 1441 125 QPGGSLRLSCAA QREFVA ESVKG SDSIRS11B121/1- 342 EVQLVESGGGLV 617 INTMG 892 WYRQAPGE 1167 EITEGGIINYTD 1442127 QPGGSLRLSCAA QRELVA SVKG SGSSAS 12D31/1- 343 EVQLVESGGGLV 618 FNDMG893 WYRQGPGK 1168 LINVGGVAKYE 1443 115 QPGGSLRLSCAA EREFVA DSVKG SRNIFD11B51/1- 344 EVQLVESGGGLV 619 GRGMG 894 WFRQAPGK 1169 AVSWSGGNTY 1444127 QAGGSLRLSCAA EREFVA YADSVKG SGGTFS 13G51/1- 345 EVQLVESGGGLV 620GRAVG 895 WFRQAPGE 1170 GISWSGGSTD 1445 127 QAGGSLSLSCAA EREFVT YADSVKGSGGTFN 13F82A/1- 346 EVQLVESGGGLV 621 GRAMG 896 WFRQAPGK 1171 FVAATSWSGG1446 130 QAGGSLRLSCAIS EREFRE SKYVADSVTG GRTLS 13E101/1- 347EVQLVESGGGLV 622 NDHMG 897 WFRQAPGT 1172 ATGRRGGPTY 1447 128QAGGSLRLSCAV ERELVA YADSVKG SGRTFN 22B85/1- 348 EVQLVESGGGLV 623 INAMG898 WYRQAPGN 1173 TITGSTGTTYA 1448 120 RPGGSLRLSCAT QRELVA DSVKG SGSDIG11B12/1- 349 EVQLVESGGGLV 624 NYAMG 899 WFRQAPGK 1174 AINWSGSHTDY 1449118 QAGGSLRLSCAA EREFVS GDSVKG SGRALI 13G61/1- 350 EVQLVESGGGVV 625SYVMG 900 WVRQAPGK 1175 GITRNSGRTRY 1450 118 QAGGSLRLSCAP AREFVA ADSVKGSGRTFS 14H41/1- 351 EVQLVESGGGLV 626 SYVMG 901 WVRQAPGK 1176GITRNSIRTRYA 1451 118 QAGGSLRLSCAP AREFVA DSVKG SGRTFS 11B81/1- 352EVQLVESGGGLV 627 NYIMG 902 WFRQALGQ 1177 AINRNGATAAY 1452 126QAGGSLRLSCAA GREFVA ADSVKG SGRPVN 11C11/1- 353 EVQLVESGGGLV 628 AYAMG903 WFRQAPGK 1178 TIRWTGGSSST 1453 121 QAGGSLRLSCAA ERESVA SYADSVKGSGRTFS 12D92/1- 354 EVQLVESGGGLV 629 MA 904 WFRQAPGK 1179 AMNWSGGSTK1454 123 QAGGSLRLSCAA EREFVA YAESVKG SGRTYN 13E61/1- 355 EVQLVESGGGLV630 MG 905 WFRQAPGK 1180 AISWSQYNTKY 1455 123 QAGGSLRLSCTA EREFVA ADSVKGSGQTFN 22B71/1- 356 EVQLVESGGAFV 631 FNVMG 906 WYRQGPGQ 1181 SITYGGNINYG1456 114 QPGGSLRLSCAA QLELVA DPVKG SGSDVW 21A121/1- 357 EVQLVESGGGLV 632MG 907 WFRQAPGK 1182 GVNWGGGSTK 1457 123 QAGGSLRLSCTA EREFVA VADSVKESGRAFN 13F101/1- 358 EVQLVESGGGLV 633 DLHMG 908 WFRQAPGK 1183FTRWPSITYIAE 1458 124 QAGGSLRLSCQL EREFVG HVKG SGGTVS 11A43/1- 359EVQLVESGGGLV 634 VNHMG 909 WYRQAPGK 1184 AITSDHITWYA 1459 123QAGGSLRLSCAA QREFVA DAVKG SGSIFR 12C81/1- 360 EVQLVESGGGLV 635 DNTMA 910WYRQAPGN 1185 TINVGGGTYYA 1460 117 QPGGSLRLSCAG QRDLVA GPVKG SGNIVR11B21/1- 361 EVQLVESGGGLV 636 MYLMG 911 WFRQAPGK 1186 TINRRGGNTYY 1461124 QAGGSLRLSCAA EREFVS ADSVKG SGRTFS 11B71/1- 362 EVQLVESGGGLV 637RYAMG 912 WFRQAPGK 1187 TISWSGGRDTV 1462 126 QAGGSLRLSCAA EREFVA YADSVKGSGRTFE 12C121/1- 363 EVQLVESGGDLV 638 YSGIG 913 WFRQAPEK 1188CIESGDGTTTY 1463 126 QPGESLRLSCAV EREAVS VDSVKG SGVTVD 22C51/1- 364EVQLVESGGGLV 639 RYDIG 914 WFRQAPGK 1189 AINWSGGTTSF 1464 121QAGASLRLSCAA GREFVA GDSVKG SGRTFS 12D11/1- 365 EVQLVESGGGLV 640 GSRMG915 WFRQAPGK 1190 AIRWSGGITWY 1465 123 QTGGSLRLSCAA EREFVA AESVKS SGRTFS12D14/1- 366 EVQLVESGGGLV 641 GSRMG 916 WLRQAPGK 1191 AVRWSGGITW 1466123 QAGGSLRLSCAA EREFVA YAESVKG SGRTFS 12C111/1- 367 EVQLVESGGGLV 642SYAMG 917 WFRQAPGK 1192 TISRSGGRTSY 1467 123 QAGGSLRLSCAV VREFVA ADSVKGSGLTFS 22B55/1- 368 EVQLVESGGGLV 643 TYAMG 918 WFRQAPGK 1193 TISRSGGRTSY1468 123 QAGGSLRLSCAV VREFVA ADSVKG SGLTFS 14H121/1- 369 EVQLVESGGGLV644 FKAMG 919 WFRQGPGK 1194 RIAGGSTNYAD 1469 113 QPGGSLRLSCAA RRELVASVKG SGITFR 12C71/1- 370 EVQLVESGGGLV 645 SYALG 920 WFRQSPGK 1195AIDWDGSRTQ 1470 125 QAGGSLRLSCTA ERESVA YADSVKG SGGTFG 21A31/1- 371EVQLVESGGGLV 646 SVAMG 921 WFRQGPGK 1196 TITWSGDSTYV 1471 125QAGGSLRLSCAA EREFAA TDSVKG SEPTFS 12C91/1- 372 EVQLVESGGGLV 647 YYHMA922 WFRQAPGK 1197 AINLSSGSTYY 1472 121 QAGGSLRLSCVA EREFIA PDSVKG SGRTSS14H81/1- 373 EVQLVESGGGLV 648 NYRMA 923 WFRQAPRK 1198 AISRSGESTYF 1473125 QAGGSLSLSCAA EREFVA ADSMKG SGRTFS 12C42/1- 374 EVQLVESGGGLV 649RYAMH 924 WFRQAPGS 1199 GISWDGGSTF 1474 124 QAGGSLRLSCAA ERDFVA YANSVKGSGRTFS 12D102/1- 375 EVQLVESGGGLV 650 FNAMG 925 WSREAPGK 1200RIISDDSTLYAD 1475 118 QPGGSLRLSCAA RRELVA SVKG SGSSLS 11A52/1- 376EVQLVESGGGLV 651 NYAMR 926 WFRQAPGK 1201 TINWSGSHTDY 1476 120QAGGSLRLSCAA EREFVA ADSVKG SGRALS 14H111/1- 377 EVQLVESGGGLV 652 SFAMR927 WFRQAPGK 1202 AINWSGTHTDY 1477 120 QAGGSLRLSCAA EREFVA ADSVKG SGRALI11B61/1- 378 EVQLVESGGGLV 653 GYGMG 928 WFRQAPGK 1203 AVGWYGSTYF 1478120 QAGGSLRLSCAA EREFVA ADSVKG SGRTSS 12E42/1- 379 EVQLVESGGGLV 654LRTMG 929 WYRQAPGN 1204 LISAGDSTYYP 1479 118 QPGGSLRLSCAH QRELVA DSVKGSGRAFS 13F81A/1- 380 EVQLVESGGGLV 655 RYAMG 930 WFRQAPGK 1205AISWTGGSSYY 1480 128 QAGGSLRLSCAA EREFVA GDSVKG SGRTFS 11B102/1- 381EVQLVESGGGLV 656 SHAIS 931 WFRQAPGK 1206 AINWSGSHRD 1481 118QAGGSLRLSCAA AREFVA YADSAKG SGGIFS 21A41/1- 382 EVQLVESGGGLV 657 NYAWS932 WFRQAPGK 1207 AINWSGGYTD 1482 120 QAGGSLRLSCAA ERGFVA YADSVKG SGRIFS14H101/1- 383 EVQLVESGGGLV 658 SSPMG 933 WFRQAPGK 1208 ATTRSGGLPYY 1483128 QAGGSLRLSCAA EREVVA SDSVKG SGRTFI 12E21/1- 384 EVQLVESGGGLV 659IHVVG 934 WYRKAPGK 1209 YIGTAGATHYA 1484 115 QPGGSLRLSCAA QREVVA DSVKGSGSIDS 13F21/1- 385 EVQLVESGGGLV 660 INATS 935 WYRQAPGN 1210 TIIGDGRTHYA1485 123 QSGGSLRLSCVA QRELVA DSVKD SGTIVS 12E33/1- 386 EVQLVESGGGMV 661NYGMG 936 WFRQAPGK 1211 SINWSGTHTYD 1486 119 QAGGSLRLSCAA EREFVS ADFVKGSGLTLS 13G11/1- 387 EVQLVESGGGLV 662 SNYAMG 937 WFRQAPGK 1212TINWSGSHSDY 1487 122 QAGGSLRLSCAA EREFVA ADSVKG SGRTFI 118N121_A6_2_OK/388 EVQLVESGGGLV 663 GYSVG 938 WFRQSPGK 1213 GINWSGRTYY 1488 1-123QAGGSLRLSCVA EREFVG VDSVKG SGRTFS 118N121_B8_1_OK/ 389 EVQLVESGGGLV 664FASYAMG 939 WFRQAPGK 1214 AIRGSGGSTYI 1489 1-135 QDGGSLRLSCAA AREFVAADPARSTYYAD SGQLAN FVKG 118N121_A2_2_OK/ 390 KVQLVESGGGLV 665 NYSVG 940WFRQAPGK 1215 ALSKDGARTYY 1490 1-124 QAGGSLRLSCAA EREFVA AASVKG SGRTFS118N121_A8_2_OK/ 391 EVQLVESGGGLV 666 SHAMG 941 WFRQAPGE 1216TIRWSGSATFY 1491 1-124 QAGGSLTLSCVIS EREFVA SDSVKG GLTLE118N121_B3_1_OK/ 392 EVQLVESGGGLV 667 DLALG 942 WFRRAPGK 1217AISSSGVTTIYA 1492 1-123 QPGGSLRLSCAA EREHVA DSVRG SGRTFS118N121_A5_2_OK/ 393 EVQLVESGGGSV 668 TNAMG 943 WHRQVSGK 1218IVTDGFTNYAD 1493 1-114 QPGGSLRLSCVA ERELVA FAKG SGSISS 118N121_A9_2_OK/394 EVQLVESGGGSV 669 VNAMG 944 WHRQVPGK 1219 IVTDGFTNYAD 1494 1-114QPGGSLRLSCVA QRELVA FAKG SGSISS 118N121_A7_1_OK/ 395 EVQLVESGGGLV 670IDVMG 945 WHRQAPGK 1220 DISFGGNTNYA 1495 1-122 QPGGSLRLSCAA ERELVS NSVKGSGNIKS 118N121_A10_1_OK/ 396 EVQLVESGGGLV 671 DYAIG 946 WFRQAPGK 1221CIANSEGTKYY 1496 1-131 QAGGSLRLSCAA EREGVS ADSAQG SGFSFA118N121_A11_1_OK/ 397 EVQLVESGGGLV 672 MYGMR 947 WVRQAPGK 1222SINSDGDTTYY 1497 1-120 QPGGSLRLSCAA GPERVS ADSVKG SGFPFG118N121_B7_4_OK/ 398 EVQLVESGGGLE 673 SAAMG 948 WFRQAPGK 1223AISRDGAATYY 1498 1-124 QAGGSLRLSCAA EREFVA TDSVKG SGLTFR118N121_B2_1_OK/ 399 EVQLVESGGGLV 674 DRAIA 949 WFRQAPGK 1224CITPHHGGIIFT 1499 1-130 QAGGSLRLSCAA AREGVS RESVKG SGFSLD118N121_B7_1_OK/ 400 EMQLVESGGGLV 675 INAMA 950 WYRQAPGN 1225 AVTSGGGTNY1500 1-119 QPGGSLRLSCAA ERELVA ATSVKG SGNIPP Clone FR3 ID CDR 3 ID FR413D11 RFTISRDNAKNTLFLQMNS 1501 GWKIVPTNP 1776 RGHGTQVTVSS LNPEDTAVYYCGQ264 RFTISRDNAKNTLFLQMNS 1502 GWKIRPTIP 1777 MGHGTQVTVSS LSPEDTAVYYCNQ2G2 RFTISRDNAKNTLFLQMNN 1503 GWKIVPTDL 1778 GGHGTQVTVSS LTPEDTAVYYCNR13D2 RFTISRDNAKNTLFLQMNN 1504 GWKIVPTDR 1779 GGHGTQVTVSS LRSEDTAVYSCNQ2D5 RFTISRDNAKNTLFLQMNS 1505 GWKIVPTDR 1780 GGHGTQVTVSS LRSEDTAVYYCNQ2F4 RFTISRDNAKNTLFLQMNS 1506 GWKIVPTDR 1781 RGHGTQVTVSS LRSEDTAVYYCNQ2C3 RFTISRDNAKNTLFLQMNS 1507 GWKIVPTDR 1782 TGHGTQVTVSS LRSEDTAVYYCNQ17E3 RFTISRDNAKNTVYLEMNS 1508 GWKIVPTDR 1783 TGHGTQVTVSS LTPEDTAVYYCNQ17H3 RFTISRDNAKNTLFLQMNS 1509 GWKIVPTDR 1784 GGHGTQVTVSS LRSEDTAVYYCNQ17D2 RFTISRDNAKNTLFLQMNS 1510 GWKIVPTDR 1785 GSHGTQVTVSS LRSEDTAVYYCNQ2F1 RFTISRDNAKNTLFLQMNS 1511 GWKIVPMDR 1786 RGHGTQVTVSS LTPEDTAVYYCNQ2E2 RFTISRDNAKNTLFLQMNS 1512 GWKIIPTDR 1787 RGHGTQVTVSS LTPEDTAVYYCNQ2C2 RFTISRDNARNTLFLQMNS 1513 GWKILPTDR 1788 RGHGTQVTVSS LTPEDTAIYYCNQ2E3 RFTISRDNAKNTLFLQMNS 1514 GWKILPTNR 1789 GSHGTQVTVSS LSPEDTAVYYCNQ13B10 RFTISRDNAKNTLFLQMNS 1515 GWKILPTNR 1790 GSHGTQVTVSS LSPEDTAVYYCNQ2D1 RFTISRDNAKNTLFLQMNS 1516 GWKILPTNR 1791 GSHGTQVTVSS LSPEDTAVYYCNR2H3 RFTISRDNAKNTLFLQMNS 1517 GWKIIPTDR 1792 RGHGTQVTVSS LTPEDTAVYYCNQ2H1 RFVISRDNAKNTLFLQMNS 1518 GWKIIPTDR 1793 RGHGTQVTVSS LSPEDTAVYYCNQ2C1 RFTISRDNAKNTLFLQMNS 1519 GWKIIPTDR 1794 RGHGTQVTVSS LTPEDTAVYYCNQ15C5 RFTISRDNAKNTLFLQMNS 1520 GWKIIPTDR 1795 RGHGTQVTVSS LTPEDTAVYYCNQ2B3 RFTISRDNAKNTLFLQMNS 1521 GWKIIPTDR 1796 RGHGTQVTVSS LTPEDTAVYYCNQ29H2 RFTISRDNAKNTLFLQMNN 1522 GWKIIPTDR 1797 RGHGTQVTVSS LTPEDTAVYYCNQ17E4 RFVISRDNAKNTLFLQMNS 1523 GWKIIPTDR 1798 RGHGTQVTVSS LSPEDTAVYYCNQ17A2 RFTISRDNAKNTLFLQMNS 1524 GWKVWPTDR 1799 GTHGTQVTVSS LSPEDTAVYYCNK15D1 RFTISRDNAKNTLFLQMNS 1525 GWKVWPTDR 1800 GTHGTQVTVSS LNPEDTAVYYCNQ17B8 RFTISRDNAKNTLFLQMNS 1526 GWKILPAER 1801 RGHGTQVTVSS LTPEDTAVYYCNQ15C11 RFTISRDNAKNTLFLQMNS 1527 GWKILPAER 1802 RGHGTPVTVSS LTPEDTAVYYCNQ15G8 RFTISRDNAKNTLFLQMNS 1528 GWKILPAER 1803 RGHGTQVTVSS LTPENTAVYYCNQ17H14 RFTISRDNAKNTLFLHMNN 1529 GWKIHPADR 1804 GGHGTQVTVSS LSPEDTAVYYCGQ27G8 RFTISRDNAKNTLFLQMNS 1530 GWKILPAER 1805 RGHGTQVTVSS LTPEDTAVYYCNQ38C6 RFTISRDNAKNTLFLQMNS 1531 GWKIRPTIP 1806 MGHGTQVTVSS LSPEDTAVYYCNQ2A4 RFTISRDNAKKTVYLQMNS 1532 GWQSTTKN 1807 WGQGTQVTVSS LQSEDTAVYYCGT QGY15G7 RFTISRDNNKKTVYLQMN 1533 GWQSTTKN 1808 WGQGTQVTVSS SLKSEDTAVYYCATQGY 15B7 RFTISRDNAKKTVYLQMNS 1534 GWQSTTKS 1809 WGQGTQVTVSSLQSEDTAVYYCGT QGY 5G4 RFTISRDNAKKTLYLQMNS 1535 GWQSTTKN 1810 WGQGTQVTVSSLKSEDTAVYYCAT QNY 13B2 RFTISRNNANNTLYLQMN 1536 NWRDAGTT 1811 AGQGTQVTVSSNLKFEDTAVYYCAK WFEKSGS 2E5 RFTISRNNANNTLYLQMN 1537 NWRDAGTT 1812AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 15G1 RFTISRNNANNTLYLQMNS 1538NWRDAGTT 1813 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 27B1 RFTISRNNANNTLYLQMN1539 NWRDAGTT 1814 AGQGTQVTVSS NLKFEDTAVYYCAK WFEKSGS 17E7RFTISRNNANNTLYLQMNS 1540 NWRDAGTT 1815 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS17D8 RFTISRNNANNMLYLQMN 1541 NWRDAGTT 1816 AGQGTQVTVSS SLKSEDTAVYYCAKWFEKSGS 5F8 RFTISRNNANNTLYLQMN 1542 NWRDAGTT 1817 AGQGTQVTVSSNLKFEDTAVYYCAK WFEKSGS 2D4 RFTISRNNANNTLYLQMNS 1543 NWGDAGTT 1818AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 13D8 RFTISRNNANNTLYLQMNS 1544 NWGDAGTT1819 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17G8 RFTISRNNANNTLYLQMNS 1545NWGDAGTT 1820 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2H4 RFTISRNNANNTLYLQMNS1546 NWGDAGTT 1821 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2F3RFTISRNNANNTLYLQMNS 1547 NWGDAGTT 1822 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS2F5 RFTISRNNANNTLYLQMNS 1548 NWGDAGTT 1823 AGQGTQVTVSS LKSEDTAVYYCAKWFEKSGS 30E10 RFTISRNNANNTLYLQMNS 1549 NWGDAGTT 1824 AGQGTQVTVSSLKSEDTAVYYCAK WFEKSGS 29H1 RFTISRNNANNTLYLQMNS 1550 NWGDAGTT 1825AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 17E2 RFTISRNNANNTLYLQMNS 1551 NWGDAGTT1826 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 2B1 RFTISRNNANNTLYLQMNS 1552NWGDAGTT 1827 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 2A5 RFTISRNNANNTLYLQMNS1553 NWGDAGTT 1828 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 13C12RFTISRNNANNTLYLQMNS 1554 NWGDAGTT 1829 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS17E10 RFTISRNNANNTLYLQMNS 1555 NWGDAGTT 1830 AGQGTQVTVSS LKSEDTAVYYCAKWFEKSGS 27D4 RFTISRNNANNTLYLQMNS 1556 NWGDAGTT 1831 AGQGTQVTVSSLKSEDTAVYYCAK WFEKSGS 15F9 RFTISRNNANNTLYLQMNS 1557 NWGDAGTT 1832AGQGTQVTVSS LKSDDTAVYYCAK WFEKSGS 30H9 RFTISRNNANNTLYLQMNS 1558 NWGDAGTT1833 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 39C1 RFTISRNNANNTLYLQMNS 1559NWGDAGTT 1834 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS 27G2 RFTISRNNANNTLYLQMNS1560 NWGDAGTT 1835 AGPGTQVTVSS LKSDDTAVYYCAK WFEKSGS 2D3RFTISRNNANNTLYLQMNS 1561 NWRDAGTT 1836 AGQGTQVTVSS LKSEDTAVYYCAK WFEKSGS5F7 RFTISRDNAKNTVYLQMN 1562 FRTAAQGTDY 1837 WGQGTQVTVSS SLKPEDTAVYYCKR118N121_A1_4_OK/ RFTISRDNAKNTLHLQMNS 1563 HKTPYTTLE 1838 WGQGTQVTVSS1-127 LKPEDTAVYYCAY LNRPHAFGS 47D5 RFTISRVNAKDTVYLQMNS 1564 DQRLDGSTL1839 WGQGTQVTVSS LKPEDTAVYYCYM AY 14B11 RFTISRDNAKKTVYLQMNS 1565ETYGSGSSL 1840 WGQGTQVTVSS LKPEDTARYYCGV MTEYDY 14B10RFTISRDNAKETVYLQMNS 1566 ETYGSGSSL 1841 WGQGTQVTVSS LKPDDTGVYYCAA MSEYDY14B4 RFTISRDNAKETVYLQMNS 1567 ETYGSGSSL 1842 WGQGTQVTVSS LKPEDTGVYYCAAMSEYDY 14C11 RFTISRDNAKETVYLQMNS 1568 ETYGSGSSL 1843 WGQGTQVTVSSLKPDDTGVYYCAA MSEYDY 14B5 RFTISRDNAKETVYLQMNS 1569 ETFGSGSSL 1844WGQGTQVTVSS LKPDDTGVYYCAA MSEYDY 14C6 RFTISRDNAKKTVYLQMNS 1570 DTYGSGSSL1845 WGQGTQVTVSS LKPEDTAVYYCAT MNEYDY 14A4 RFTISRDNAKKTAYLQMNS 1571ETYGSGSSL 1846 WGQGTQVTVSS LKPEDTAVYYCAA MNEYDY 14B3 RFTISRDNAKKTAYLQMNS1572 ETYGSGSSL 1847 WGQGTQVTVSS LKPEDTAVYYCAA MNEYDY 14C1RFTISRDNAKKTVYLQMNS 1573 ETYGSGSSL 1848 WGQGTQVTVSS LKPEDTAVYYCAT MNEYDY14A12 RFTISRDNAKKTVYLQMNS 1574 EPYGSGSSL 1849 WGHGTQVTVSS LKPEDTAVYYCAAISEYDY 14A2 RFTISRDNAKKTVYLQMNS 1575 EPYGSGSSL 1850 WGHGTQVTVSSLKPEDTAVYYCAA ISEYDY 14A1 RFTISRDNAKKTVYLQMNS 1576 EPYGSGSSL 1851WGHGTQVTVSS LKPEDTAVYYCAA MSEYDY 17C3 RFTMARDNANHTVHLQM 1577 NWKMLLGV1852 WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 46D3 RFTVSRDIAENTVYLQMNT 1578KIGWLSIRG 1853 WGQGTQVTVSS LKSEDTAVYYCAA DEYEY 27H5 RFTISSDNAKNTVYLQMNS1579 LPFVCPSGS 1854 WGQGTQVTVSS LKPEDTAVYYCAA YSDYGDEY DY 17C2RFTISRDNAKNTLYLQMSS 1580 HVSDSDYTE 1855 WGQGTQVTVSS LKPEDTALYYCTK YDY17D11 RFTISRDNAKNTLYLQMNS 1581 RRSSLYTSS 1856 WGQGTQVTVSS LKPEDTAVYYCAANVFEYDY 15A6 RFTVSRDNANNTVYLQMN 1582 VNADY 1857 WGQGTQVTVSSTLKPEDTAVYYCRD 17B6 RFTVSRDNAKNTVYLQMN 1583 VNADY 1858 WGQGTQVTVSSSLKAEDTAVYYCWD 17C5 RFTVSRDNANNTVYLQMN 1584 VNADY 1859 WGQGTPVTVSSTLKPEDTAVYYCRD 15E11 RFTVSRDNDKNTLYLQMN 1585 VNADY 1860 WGQGTQVTVSSSLKPEDTAVYYCRD 15C2 RFTVSRDNANNTVYLQMN 1586 VNADY 1861 WGQGTQVTVSSTLKPEDTAVYYCRD 2A3 RFTVSRDNAKNTLYLQMN 1587 VNGDI 1862 WGQGTQVTVSSSLKPEDTATYLCRD 27A5 RFTVSRDNAKNTLYLQMN 1588 VNGDI 1863 WGQGTPVTVSSSLEPEDTATYYCWD 2C5 RFTVSRDNAKNTLYLRMN 1589 VNADI 1864 WGQGTQVTVSSSLKSEDSATYTCRA 27G5 RFTVSRDNAKNTVYLQMN 1590 VNGDY 1865 WGQGTQVTVSSSLKPEDTAVYYCRD 13A9 RFTVSRDNANHTVYLQMN 1591 VNGDY 1866 WGQGTQVTVSSSLKPEDTAVYYCRD 29E9 RFTVSRDNAKNTVYLQMN 1592 VNGDY 1867 WGQGTQVTVSSSLKPEDTAVYYCRD 15D8 RFTVSRDNAKNTVYLQMN 1593 VNGDY 1868 WGQGTQVTVSSSLKPEDTAVYYCRD 15G4 RFTVSRDNAKNTLYLQMN 1594 VNGDY 1869 WGQGTQVTVSSSLKPEDTAVYYCWD 15D12 RFAISRDNAKNTAYLQMN 1595 VWKLPRFV 1870 WGQGTQVTVSSSLKAEDTAVYYCNA DNDY 15E12 RFTMARDNANHTVHLQM 1596 NWKMLLGV 1871WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 13D7 RFTMARDNANHTVHLQM 1597 NWKMLLGV1872 WGQGTQVTVSS NSLKPEDTAVYYCNA ENDY 13A8 RFTISRDNAENTVYLQMN 1598 VNRDY1873 WGQGTQVTVSS SLKPEDTAVYYCWD 15A4 RFTISRDNAKNTVYLQINS 1599 VNRDY 1874WGQGTQVTVSS LKPEDTAVYYCWD 17F7 RFTISRDNAKKTMYLQMN 1600 VNRDY 1875WGQGTQVTVSS SLKPDDTAVYYCRD 15C8 RFTVSRDNAENTVYLQMN 1601 VNRDY 1876WGQGTQVTVSS SLKPEDTAVYYCRD 17A10 RFTLSRDNAKNTVYLQMN 1602 VNRDY 1877WGQGTQVTVSS SLKPEDTAVYYCRD 27D3 RFTISRDNAENTVYLQMN 1603 VNRDY 1878WGQGTQVTVSS SLKPEDTAVYYCWD 13B12 RFTLSRHNAENTVYLQMN 1604 VNRDY 1879WGQGTQVTVSS SLKPEDTAVYYCRD 15B2 RFTISSDNAKSTIYLQMNS 1605 VNRDY 1880WGQGTQVTVSS LKPDDTAVYYCRD 15B11 RFTISSDNAKNTIYLQMNS 1606 VNRDY 1881WGQGTQVTVSS LKPDDTAVYYCRD 13C9 RFTISRDNAKKTVYLQMNS 1607 VNRDY 1882WGQGTQVTVSS LKPEDTAVYYCRD 17D5 RFTISRDGATNTVFLQMN 1608 VNRDY 1883WGQGTQVTVSS NLKPEDTAVYYCRD 27B5 RFTVSRDNAENTVYLQMN 1609 VNRDY 1884WGQGTQVTVSS SLKPEDTAVYYCRD 27C7 RFTVSRDNADNTVYLQMN 1610 VNRDY 1885WGQGTQVTVSS SLKPEDTAVYYCRD 13D4 RFTISSDNAKSTIYLQMNS 1611 VNREY 1886WGQGTQVTVSS LEPDDTAVYYCRD 15G5 RFTISSDNAKKTIYLQMNS 1612 VNREY 1887WGQGTQVTVSS LKPDDTAVYYCRD 13C4 RFTISSDNAKSTIYLQMNS 1613 VNREY 1888WGQGTQVTVSS LKPDDTAVYYCRD 46G1 RFTISRDNAKNAVILQMNN 1614 KPGWVARD 1889WGQGTQVTVSS LKTEDTAVYYCAA PSQYNY 46E4 RFTISRDNAKNAVILQMNN 1615 KPGWVARD1890 WGQGTQVTVSS LKTEDTAVYYCAA PSEYNY 17B5 RFTISNDNAKDTVYLQMN 1616 FNRDL1891 WGQGTQVTVSS SLKSEDTAVYYCRN 15C9 RFTISRDNAKNTVYLQMN 1617 VNRDL 1892WGQGTQVTVSS SLKPEDTGVYYCRD 13D10 RFTISKDYTRNTVYLQVNS 1618 DANPNCGL 1893WGQGTQVTVSS LKPEDTAVYHCAT NVWNS 17C6 RFTISGDTAKSTLYLQMNS 1619 RDYYYMPF1894 WGQGTQVTVSS LKPEDTAMYYCYG 15A2 RFAISRDNAKNTVYLQMN 1620 INEDQ 1895WGQGTQVTVSS SLKPEDTAVYYCRD 17A8 RFAISRDNAKNMVYLQMN 1621 INEDQ 1896WGQGTQVTVSS TLKPEDTAVYYCRD 15G10 RFAISRDNAKNTVYLQMN 1622 INEDQ 1897WGQGTQVTVSS SLKPEDTAVYYCRD 27A3 RFAISRDNAKNTVYLQMN 1623 INEDQ 1898WGQGTQVTVSS TLKPEDTAVYYCRD 17H10 RFAISRDNAKNTVYLQMR 1624 INADQ 1899WGQGTQVTVSS SLKPEDTAVYYCRD 30D10 RFTISRDNAKNTVYLQMN 1625 INRDI 1900WGQGSQVTVSS SLKPEDTAVYYCRD 15H4 RFTISRDNAKNTVYLQMN 1626 INHDI 1901WGRGSQVTVSS NLKPEDTAVYYCRD 17B7 RFTISRDNAKKTVYLQMNS 1627 INRDY 1902WGQGTQVTVSS LKPEDTAVYFCRD 15D2 RFTFSKDGAKNTVYLQMN 1628 INRDY 1903WGQGTQVTVSS SLKPEDTAVYYCRD 17G5 RFTISRDNAKNTVYLQMN 1629 INRDY 1904WGQGTQVTVSS SLKPEDTAVYYCRD 15B6 RFTLSRDNAKKTVYLQMN 1630 INGDY 1905WGQGTQVTVSS SLKPEDTAIYYCRD 27F2 RFTISISRDNVEYTVDLQM 1631 INGDY 1906WGQGTQVTVSS DSLKPEDTAVYYCRD 17F5 RFTISRDNAKNTVYLQVNS 1632 TNGDY 1907WGQGTQVTVSS LKPEDTAVYYCWD 17B2 RFTISRDNAKNTLYLQMNS 1633 DISTFGWGP 1908WGQGTQVTVSS LKPEDTALYYCTK FDY 27H4 RFTISRDNAKNTVYLEMNS 1634 RWDIVGAIW1909 WGQGTQVTVSS LKPEDTGVYYCNG 13A4 RFTISRDNAKNTVYLEMNS 1635 RWDIVGAIW1910 WGQGTQVTVSS LKPEDTGVYYCNG 2A1 RFTISRDNTKNTLDLQMNS 1636 RWLEIGAEY1911 WGQGTQVTVSS LKPEDTAVYYCHG 15E10 RFTISRDNAKNTVVLQMN 1637 SWSYPGLTY1912 WGKGTLVTVSS GLKPEDTAVYYCKG 27E7 RFTISRDNAKNTVYLQMN 1638 VWRAIGRTY1913 WGQGTQVTVSS DLKPEDTAVYYCNG 47E5 RFTIARDNAKNTVSLQMN 1639 DRRSYNGR1914 WGQGTQVTVSS SLKPEDTAVYYCNL QY 2G4 RFTISRDNTKNTMYLQMV 1640 GGREYSGV1915 WGQGTQVTVSS SLKPEDTAVYYCNV YYYREY 14D4 RFTISRDNAKNTVYLQMAS 1641RPSDTTWS 1916 WGQGTQVTVSS LKPEDTAAYYCAA ESSAS 17A5 RFTISRDNVKNTLYLQMNS1642 DLGNSGRG 1917 WGQGTQVTVSS LKSEDTAVYYCAK PYTN 15D10RFTISRDNAKNTVYLQMN 1643 DCGVPGFG 1918 WGQGTQVTVSS SLKPEDTAVYYCAT WTFSS13C2 RFTISRDNAKNAVHLQMN 1644 KQLPYLQNF 1919 WGQGTQVTVSS SLRLEDTAVYYCNA17G11 RFTISRDNPETAVHLQMN 1645 KQLPYLQNF 1920 WGQGTQVTVSS SLKLEDTAVYYCNA17A3 RFTISRDNARDTLFLQMHA 1646 HRWGLNY 1921 WGQGTQVTVSS LQPEDTAVYMCNL27B7 RFTISRDNAKNTLYLQMSS 1647 ARSSSSYYD 1922 WGQGTQVTVSS LKPEDTALYYCAKFGS 17A6 RFTISTDNAKNTLYLQMSS 1648 ARSSSSYYD 1923 WGQGTQVTVSSLKPEDTALYYCAK FGS 17D7 RFTISRDNAKNTVYLQMN 1649 DRGSGTCY 1924 WGQGTQVTVSSSLKPEDTAVYYCAT ADFGS 46D4 RFTISRDNAKKTLYLQMNS 1650 GWGPAVTSI 1925ATLGTQVTVSS LQSEDTAVYYCAK PV 27B3 RFTISRDNAKNTVYLQMN 1651 INRDI 1926WGQGSQVTVSS SLKPEDTAVYYCRD 27E5 RFTISRDNAKNTVYLQMN 1652 INRDI 1927WGQGSQVTVSS SLKPEDTAVYYCRD 27D6 RFTISRDNAKNTVYLQMN 1653 INRDI 1928WGQGSQVTVSS SLKPEDTAVYYCRD 30D10 RFTISRDNAKNTVYLQMN 1654 INRDI 1929WGQGSQVTVSS SLKPEDTAVYYCRD 47G11 RFTISRDNAKNTVYLQMN 1655 SRDYSRSR 1930WGQGTQVTVSS SLKPEDTAVYYCAS DPTSYDR 27C3 RFTISRDNAKNTLYLQMNS 1656PRGSSLYLL 1931 WGQGTQVTVSS LKPEDTAVYYCAR EYDY 11A101/1-RFTTSRDNAKNTVYLQMN 1657 DFYLATLAH 1932 WGQGTQVTVSS 120 DLKPEDTAVYYCAAEYDY 11A22/1- RFTISRDNAKNTVYLQMN 1658 DFYVSTLAH 1933 WGQGTQVTVSS 122SLKPEDTAVYYCAA EYDY 12D44/1- RFTISRANAKNTVYLQMN 1659 DFYVSTLAH 1934WGQGTQVTVSS 122 GLKPEDTAVYYCAA EYDY 12E11/1- RFTISRDNAKITVYLQMNS 1660DFYVSTLAH 1935 WGQGTQVTVSS 122 LKPEDTAVYYCAA EYDY 13G111/RFTISRDNAKNTVYLQMN 1661 DTFTLSTLS 1936 WGQGTQVTVSS 1-123 SLKPEDTAVYYCAAHEYDY 13F71/1- RFTISRDNAKNTVYLQMN 1662 DLIVATLPGE 1937 WGQGTQVTVSS 123SLKPEDTAVYYCAA YDY 14H61/1- RFTISRDNAKNTVYLQMN 1663 DEILATLPHE 1938WGQGTQVTVSS 122 SLSPEDTAVYYCAA YDY 22B12/1- RFTISRDNAKNTVYLQMN 1664DAYTVIATLP 1939 WGQGTQVTVSS 124 SLKPEDTAVYYCAA HEYDY 14H71/1-RFTISRDSAKNAVNLQMEL 1665 KHSTVAGLT 1940 WGQGTQVIVSS 123 VESDDTAVYYCAAHEYDY 12D51/1- RFTISRDSAKNTVYLQMDS 1666 VYVSTWGN 1941 WGQGTQVTVSS 120LKPEDTAVYVCNA GYDY 11A111/1- RFTISRDNAKNSVYLQMN 1667 RRSGYYSLS 1942WGQGTQVTVSS 126 SLKPEDTAIYYCAA TSPHQYAY 13G71/1- RFTISRDNAKNTVYLQMN 1668RVNYAAYSR 1943 WGQGTQVTVSS 124 SLKPEDTAVYYCGA LEHDYHY 13G74/1-RFTISRDNAKNTVYLQMN 1669 RVNYAAYSR 1944 WGQGTQVTVSS 125 SLKPEDTAVYYCGALEHDYHY 11A71A/ RFTIARDDAKNTMYLQMN 1670 NRAGIYEY 1945 WGQGTQVTVSS 1-116GMKPEDTAVYYCKM 22B101/1- RFDISRDNAKNTVYLQMN 1671 RLFMATPNQ 1946WGQGTQVTVSS 123 RLKPEDTAVYYCAA GQYYY 11B42/1- RFAISRDNALNTVYLQMNS 1672RPSYSTNNV 1947 WGQGTQVTVSS 123 LKPEDTAVYYCAA KSYRY 13E111/1-RFTISRDNGKNTVDLRMN 1673 RPAVVHTRK 1948 WGQGTQVTVSS 124 SLKPEDTAVYYCAAESYPY 14H12/1- RFTISRDNAKNTVYLQMN 1674 RINYSVLTTT 1949 WGQGTQVTVSS 125SLKPEDTAVYYCAA SSSYHY 13G101/ RFTISRDDAKNTVYLQMN 1675 DFRSTWLSS 1950WGQGTQVTVSS 1-123 SLKAEDTAIYYCAA SGSSYTY 13G41/1- RFTVSRDNAQNTVYLQMN1676 RRIVNVEGA 1951 WGQGTQVTVSS 121 SLKIEDTGVYYCNA YRDY 22B910/1-RFTISGDDARNTVYLQMN 1677 KVVVAVEGA 1952 WGQGTQVTVSS 121 SLNPEDTAVYYCNAKYDY 21A81/1- RFTISRDNSENTVYFEMG 1678 CRISWCAGA 1953 WGQGTQVTVSS 122SLKPEDTAVYYCAI ESDYGY 21A92/1- RFTISRDNAKGTVYLQMS 1679 DSITDRRSV 1954WGQGTQVTVSS 127 SLKPEDTAVYYCAA AVAHTSYYY 22C712/1- RFTISRDNTKNTVYLQMNS1680 RLSSVAVAS 1955 WGQGTQVTVSS 123 MRPEDTAVYYCAA TRYDY 11A13/1-RFTISRDIANDVVYLQMNS 1681 NRQSGVAS 1956 WGQGTQVTVSS 125 LEPEDTAVYYCARENLRLYTY 13G93/1- RFTISRDNAKNTLYLQTTS 1682 DKQSGVSV 1957 WGQGTQVTVSS 123LRPEDTGVYYCAE NPKYAY 12C52/1- RFTISKDNAKNTVYLQMNS 1683 QTLGRSLYD 1958WGQGTQVTVSS 118 LKPEDTAVYYCAA Y 12C61/1- RFTISRDNAKNTVYLQMN 1684SRYSGGVA 1959 WGQGTQVTVSS 126 SLKPEDTAVYYCAA TARRSEYHY 21A61/1-RFTISRDYAKNTWYLQMN 1685 VATYSRNVG 1960 WGQGTQVTVSS 125 TLKPEDTAAYYCAASVRNYDY 11A121/1- RFTISRDNAKNTVQLQMN 1686 SQPLNYYTY 1961 WGQGTQVTVSS 126SLKPEDTAVYYCAV YDARRYDY 11A91/1- RFTISRDNAKNTVYLQMTS 1687 TPVYYQRYY 1962WGQGTQVTVSS 124 LMPEDTAVYYCAD DQNAYDY 13G72/1- RFTISRDNAKNTVYLQMN 1688KYYSYYAYD 1963 WGQGTQVTVSS 118 SLKPEDTAVYYCAA Y 13E81/1-RFTISRDNAKDTMYLQMN 1689 SPVYYIDYS 1964 WGQGTQVTVSS 124 VLKPEDTAVYYCAASQYKYGY 11B31/1- RFTISRDNAKNTVYLRMNS 1690 QIYYRTNYY 1965 WGQGTQVTVSS 124LKPEDTAVYYCAT SQNAYDY 13G81/1- RFTISRDDAKNTVYLQMN 1691 QIYYRTNYY 1966WGQGTQVTVSS 124 SLKPEDTAVYLCAA SQNNYDY 21A53/1- RVTISRDDAKNTVYLRMN 1692QIYYRTNYY 1967 WGQGTQVTVSS 124 SLKPEDTAVYYCAA SQNVYDY 14H51/1-RFTISRDDAKNTVYLRMNS 1693 QIYYRTNYY 1968 WGQGTQVTVSS 124 LKPEDTAVYYCAASQNEYDY 21A21/1- RFTISRDNAKNTVYLRMNS 1694 QIYYRTNYS 1969 WGQGTQVTVSS 124LKPEDTAVYYCAA SQSNYDY 21A111/1- RFTIARDDAKNTVYLQMN 1695 ATAYRTNYS 1970WGQGTQVTVSS 124 SLKPEDTAVYYCAL SRDKYDY 22B1212/ RFTISRDNAKNTLYLQMNS 1696YLSFYSDYE 1971 WGQGTQVTVSS 1-122 LKPEDTAVYYCAK VYDY 11A31/1-RFTISRDNAKNGVSLQMD 1697 DPTYGSGR 1972 WGQGTQVTVSS 120 SLKAEDTAVYYCAS WTY13E51/1- RFTISSDNAKNTVYLQMNS 1698 SVTYYSGSH 1973 WGQGTQVTVSS 128LKPEDTALYYCAR AYTQEGGY AR 12D121/1- RFTISRDDAKNTVYLQMN 1699 RQPYASGS1974 WGQGTQVTVSS 126 SLKPEDTAVYYCAA HYSSTQYTY 13F121/1-RFTISIDNAKNTVYLQMNN 1700 LYRGRSVYD 1975 WGQGTQVTVSS 119 LTPEDTAVYYCAA D13G121/ RFTISRDNAKNTVYLQMD 1701 SSYGSTYYS 1976 WGQGTQVTVSS 1-127SLKPEDTAVYYCAA QGRAYYYD Y 22B41/1- RFTISKENAKNTVYLQMTIL 1702 SPYGPLYRS1977 WGQGTQVTVSS 124 KPEDTAVYYCAS THYYDY 12D71/1- RFTISRDSAKDTVYLQMN1703 TTYYSGSYI 1978 WGQGTQVTVSS 125 NLKPEDTAVYYCAA STLSTSYNY 13F42/1-RFTISRDNAKNAVNLQMS 1704 GSY 1979 RGQGTQVTVSS 111 NLKPEDTALHYCTI12C101/1- RFTISRDNARNAVNPQMN 1705 GSY 1980 RGQGTQVTVSS 111NLKPEDTAVYYCTI 14H91/1- RFTISRDNAKNAVYLQMN 1706 RDSSTLDST 1981WGRGTQVTVSS 127 SLKPEDTAVYYCAA YYVGGSYN Y 13F41/1- RFTISRDNAKNAVNLQMS1707 GSY 1982 RGQGTQVTVSS 111 NLKPEDTALYYCTI 14H21/1-RFTISRSIAENTVYLQMNK 1708 RMYGSDWL 1983 WGQGTQVTVSS 125 VKPEDTAVYYCAAPRPEDFDS 22B610/1- RFTISRDNAKNTVYLQMN 1709 DVSPSYGS 1984 WGQGTQVTVSS 120SLKPEDTAVYYCNA RWYG 12C32/1- RFTISRDNAKSTMYLQMN 1710 RREYSTIYT 1985WGQGTQVTVSS 127 SLKPEDTAVYYCAA ARYPGEYVY 12D61/1- RFTISRDNAKNTLYLQMNS1711 RERGIYDS 1986 WGQGTQVTVSS 116 LKAEDTAVYYCNA 13G31/1-RFTISRDDAKNSIYLQMNT 1712 DDYLGGDN 1987 WGQGTQVTVSS 125 LKPEDTAVYYCAAWYLGPYDS 22C65/1- RFTISRDNAKNTVYLQMN 1713 RKTYRSLTY 1988 WGQGTQVTVSS 124SLIPEDTAVYYCAA YGEYDS 11A71/1- RFTVSRDNGRNTVYLLQMN 1714 DRLLYYSSG 1989WGQGTQVTVSS 125 SLKPEDTAVYYCNV YYQTSVDV 11B91/1- RFTISRDKAKNTVLLQMDS1715 DRFLYYSAG 1990 WGQGTQVTVSS 125 LKPEDTAVYYCNA RYDTGSDI 11A81/1-RFTISRDKAKNALYLQMNS 1716 DRVLYYSDS 1991 WGQGTQVTVSS 125 LKPEDMAVYYCNARYYTGSNY 11B121/1- RFTISRDNAKNTVYLEMNN 1717 DRALYRNYS 1992 WGQGTQVTVSS127 LKPEDTAVYYCNA DGRYYTGY DY 12D31/1- RFTISRDNAENTVYLQMN 1718 RILSRNY1993 WGQGNQVTVSS 115 NLKPEDMAVYYCNA 11B51/1- RFTISRDNAKSTVYLQMDS 1719SRRFYSGLY 1994 WGQGTQVTVSS 127 LKPEDTAVYYCAA YYTDDAYEY 13G51/1-RFTISRDNSKNTVSLQMN 1720 SRRFYSGLV 1995 WGQGTQVTVSS 127 SLKPEDTAVYYCAAYYSVDAYEN 13F82A/1- RFTIFRDNAENTAYLQMNS 1721 TKRYYSIKY 1996 WGQGTQVTVSS130 LNPEDTAVYYCAV YSTVEDYEY 13E101/1- RFTISRDNAESTVYLQMNS 1722 NRYYCSTY1997 WGQGTQVTVSS 128 LKAEDTAVYYCAA GCLSTPRQY DY 22B85/1-RFAISRDGAKNTVYLQMD 1723 RVYTGTYG 1998 WGQGTQVTVSS 120 SLKPEDTAVYYCNLGRNY 11B12/1- RFAISRDNAKNTVYLQMH 1724 GYSLPAFDS 1999 WGPGTQVTVSS 118SLKPEDTAVYHCAT 13G61/1- RFTISRDNADNTVTLQMN 1725 GIDLYTFHY 2000FGQGTQVTVSS 118 SLKPEDTAVYYCAG 14H41/1- RFTISRDNADNTVTLQMN 1726GIDLYTFDY 2001 FGQGTQVTVSS 118 SLKPEDTAVYYCAG 11B81/1-RFTISRDNAEDLLYLQMNL 1727 NSDSGFDS 2002 WGQGTQVTVSS 126 LKPEDTAVYYCAAYSVWAAYEY 11C11/1- RFTISKNTAENTVYLQMNS 1728 LLTVWDTYKY 2003 WGQGTQVTVSS121 LKPEDTAVYYCAV 12D92/1- RFTISRANDNNPLYLQMNT 1729 TNRWYTGV 2004WGQGTQVTVSS 123 LKPEDTAVYYCAA YDLPSRYEY 13E61/1- RFTISRDNAINSLYLQMDT1730 TNRWFSAV 2005 WGQGTQVTVSS 123 LKPEDTAVYYCAA YDLPSRYTY 22B71/1-RFSISRDNALKTVYLQMNS 1731 DLPSRL 2006 WGQGTQVTVSS 114 LKPEDTAVYYCYA21A121/1- RFTISRDYDNSPVYLQMN 1732 TSRWYSAV 2007 WGQGTQVTVSS 123TLKPEDTAVYYCAA YDLPTRYDY 13F101/1- RFTISRDNAKNTVYLQMN 1733 DRSYSIDYR2008 WGQGTQVTVSS 124 SLEREDTAVYYCAA HPDSYSY 11A43/1- RFTISRDNAKNTVTLQMNS1734 DPLLFYGVG 2009 WGQGTQVTVSS 123 LRPEDTAVYYCAA SADVDY 12C81/1-RFTISRDNAKNSVYLQMN 1735 ISGLVQRDY 2010 WGQGTQVTVSS 117 SLKPEDTSVYYCNV11B21/1- RFTISRDNARNIVYLQMN 1736 GGHLLGYD 2011 WGQGTQVTVSS 124SLKPEDTAVYYCAA VQWEPDY 11B71/1- RFTISRDNAKNIVYLQMN 1737 HKRTYELGA 2012WGQGTQVTVSS 126 SLKPEDTAVYYCAA HSTDFGS 12C121/1- RFTISRDNAKNAVYLQMN 1738AVFVDSGDF 2013 WGQGTQVTVSS 126 SLKPEDTGVYYCAT SVCRGVGY 22C51/1-RFTISRDNAKNTVYLQMN 1739 LRSWPRGV 2014 WGQGTQVTVSS 121 SLKPEDTAVYYCAADSGS 12D11/1- RFTISRDNTKNTIDLQINSL 1740 DVIYKNIGS 2015 WGQGTQVTVSS 123KPEDTAVYYCAA GSFDY 12D14/1- RFTISRDNTKNTIDLQINSL 1741 DVIYKNIGS 2016WGQGTQVTVSS 123 KPEDTAVYYCAA GSFDY 12C111/1- RFIVSRDNAKNTADLQMN 1742SKWYGGFG 2017 WGQGTQVTVSS 123 DLKPEDTAVYYCGA DTDIEY 22B55/1-RFIVSRDNAKNTADLQMN 1743 SKWYGGFG 2018 WGQGTQVTVSS 123 ELKPEDTAVYYCGADTDIEY 14H121/1- RFTISRDDAKNTVFLQMNS 1744 DGPFGN 2019 WGQGTQVTVSS 113LKPEDTAVYYCNV 12C71/1- RFTISRENVKDTMYLQMN 1745 SRHSGNTLS 2020WGQGTQVTVSS 125 SLQAEDTGVYYCVR FSLKYDY 21A31/1- RFTISRDNARNTAYLQMD 1746RRWSGTLS 2021 WGQGTQVTVSS 125 SLRPEDTAVYSCAA LFDNEYYY 12C91/1-RFTISRGNAKNTVNLQMN 1747 DNYRDSYLE 2022 WGQGTQVTVSS 121 SLKPEDTAVYYCAAYDY 14H81/1- RFTISRDNTESTGYLQMN 1748 SWDHGDYV 2023 WGQGTQVTVSS 125NLKPEDTAVYYCAA DGGFFYDY 12C42/1- RFTISRDNAKNMVYLQMN 1749 AGSAGPPSI 2024WGQGTQVTVSS 124 SLKPEDTAVYYCAA DRQYDY 12D102/1- RFTISRDYAKNTAYLQMNS 1750DVRDSIWRSY 2025 WGQGTQVTVSS 118 LKPEDTAVYYCVA 11A52/1-RFTISRDNAENTVYLQMN 1751 GWGATQAQ 2026 WGQGTQVTVSS 120 SLTPEDTAVYYCAS SGF14H111/1- RFTISRDNAENTVYLLMNS 1752 GWGATQAQ 2027 WGQGTQVTVSS 120LIPEDTAVYYCAT HGF 11B61/1- RFTIYRDNAQNTMYLQMN 1753 SSSLATISQ 2028WGQGTQVTVSS 120 SLKPEDTAVYYCAA PSS 12E42/1- RFTVSRDNAKNTVYLQMN 1754KAVTSRDHEY 2029 WGQGTQVTVSS 118 SLKPEDTAVYYCNA 13F81A/1-RSTISRENAENTVYLQMN 1755 NSDEFYSGT 2030 WGQGTQVTVSS 128 SLKPEDTAVYYCAALKLQSRMVEY 11B102/1- RFTISRDNAKKTAYLLIMNS 1756 GWKTDEYVK 2031WGQGTQVTVSS 118 LRPEDTAVYYCVG 21A41/1- RFTISRDNTKNTVYLQMNS 1757 GWVTPSYE2032 WGQGTQVTVSS 120 LKPEDTAVYYCRP YGN 14H101/1- RFTISRDNAKNTVDLQMS 1758DQKYGMSY 2033 WGQGTQVTVSS 128 SLKPEDTAAYYCAA SRLWLVSEY EY 12E21/1-RFTISRDNAENLVYLQMN 1759 GWGDSAY 2034 WGQGTQVTVSS 115 NLKPEDTAVYYCSA13F21/1- RFTISRDAAANLVYLQMNS 1760 NGIESYGW 2035 WTVGTQVTVSS 123LKPSDTAIYSCNA GNRHFNY 12E33/1- RFIISRDNAKNTVYLQINSL 1761 GGWGTGRY 2036WGVGTQVTVSS 119 KPEDTAVYYCAA NY 13G11/1- RFTISRDNAKNTVYLQMN 1762GWGTAPLS 2037 WGQGTQVTVSS 122 NLKSEDTAVYYCAP TSVY 118N121_A6_2_OK/RFTFSRDNAKNTVYLQMN 1763 DRFNTIANL 2038 WGQGTQVTVSS 1-123 SLKPEDTAIYLCAVPGEYDY 118N121_B8_1_OK/ RFTISRDNAKNTVYLQMN 1764 ETFNSISNLP 2039WGQGTQVTVSS 1-135 SLKPEDTAVYYCAC GEYDY 118N121_A2_2_OK/RFTIYRDNAKNWYLQMS 1765 DHFTFMSNL 2040 WGQGTQVTVSS 1-124 VLNGEDTAVYYCAAPSEYDY 118N121_A8_2_OK/ RFTISRDNAKNTVYLQMN 1766 RKIYRSLSY 2041WGQGTQVTVSS 1-124 SLKPEDTAVYYCAA YGDYDS 118N121_B3_1_OK/RFTISRDEAKNTVYLEMNS 1767 RLTMATPNQ 2042 WGQGTQVTVSS 1-123 LKTDDTAVYYCAASQYYY 118N121_A5_2_OK/ RFTISRDNAKTIVYLQMNS 1768 SGIGTDN 2043 WGQGIEVTVSS1-114 LQPEDTARYYCRY 118N121_A9_2_OK/ RFTISRDNAKTTVYLQMNS 1769 SGIGTDN2044 WGQGIEVTVSS 1-114 LQPEDTARYYCRY 118N121_A7_1_OK/ RFTISRDNAKNTVYLQMN1770 DILYKTDIYY 2045 WGQGTQVTVSS 1-122 SLKPEDTAVYYCYA RNDF118N121_A10_1_OK/ RLPISSDNAKKTVYLQMDS 1771 LPYTICPVVV 2046 WGKGTQVTVSS1-131 LKPEDTAVYYCAA KKGAVYYG VDDY 118N121_A11_1_OK/ RFTISRDNDENMLYLQMN1772 GFSDRSFAV 2047 KGQGTQVTVSS 1-120 SLKPEDTAVYYCAT TH 118N121_B7_4_OK/RFTISRDNAKNTVFLQMNS 1773 DFRLARLRV 2048 WGQGTQVTVSS 1-124 LKPEDTAIYYCAAADDYDY 118N121_B2_1_OK/ RFATSSDSAKNTVYLQMH 1774 LRTDYSINW 2049WGQGTQVTVSS 1-130 SLKPEDTAVYYCAT ANCQRDSL YGY 118N121_B7_1_OK/RFIISRDDSKNTVDLQMNS 1775 GGWTRTHP 2050 WGQGTQVTVSS 1-119 LKPEDTAVYYCNLFDY

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1;or from the group of CDR 1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to HER2 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable A-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table A-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table A-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table A-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table A-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table A-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table A-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table A-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table A-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table A-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table A-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table A-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table A-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable A-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e.those mentioned on the same line in Table A-1) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table A-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table A-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table A-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table A-1 (i.e.mentioned on the same line in Table A-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableA-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table A-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable A-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table A-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table A-1; a CDR2sequence, and one of the CDR3 sequences listed in Table A-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table A-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table A-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table A-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table A-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed inTable A-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequencelisted in Table A-1 that belongs to the same combination; and a CDR3sequence mentioned in Table A-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable A-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable A-1 that belongs to the same combination; and the CDR3 sequencementioned in Table A-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues: and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 2051-2325.

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring Nanobodies (from any suitablespecies), naturally occurring V_(HH) sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic amino acid sequencesor Nanobodies, including but not limited to partially humanizedNanobodies or V₁ sequences, fully humanized Nanobodies or V_(HH)sequences, camelized heavy chain variable domain sequences, as well asNanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR) to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 2051-2325.This degree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said Nanobody and one or more of the sequences of SEQ IDNO's: 2051-2325, in which the amino acid residues that form theframework regions are disregarded. Such Nanobodies can be as furtherdescribed herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 2051-2325 or from the group consisting offrom amino acid sequences that have more than 80%, preferably more than90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with at least one of the amino acidsequences of SEQ ID NO's: 2051-2325.

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 2051-2325, thatcomprise, compared to the corresponding native V_(HH) sequence, at leastone humanizing substitution (as defined herein), and in particular atleast one humanizing substitution in at least one of its frameworksequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of atleast one Nanobody of the invention. Some preferred, but non-limitingexamples of polypeptides of the invention are given in SEQ ID NO's:2051-2390.

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for HER2. Such multivalent constructs will beclear to the skilled person based on the disclosure herein: somepreferred, but non-limiting examples of such multivalent Nanobodyconstructs are the constructs of SEQ ID NO's: 2326-2390.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as“multispecific constructs”, and these may provide certain advantagescompared to the corresponding monovalent Nanobodies of the invention (aswill become clear from the further discussion herein of some preferred,but non-limiting multispecific constructs). Such multispecificconstructs will be clear to the skilled person based on the disclosureherein; some preferred, but non-limiting examples of such multispecificNanobody constructs are the constructs of SEQ ID NO's: 2331-2390.

A multispecific polypeptide or protein comprising or essentiallyconsists of at least one Nanobody of the invention and at least oneother binding unit directed against another epitope or antigenicdeterminant on HER2 (which is preferably also a Nanobody) is alsoreferred to as a “multiparatopic” protein or polypeptide or a“multiparatopic construct”.

Some preferred, but non-limiting examples of bivalent monospecificpolypeptides of the invention are given in SEQ ID NO's: 2326-2330. Somepreferred, but non-limiting examples of bispecific polypeptides of theinvention are given in. SEQ ID NO's: 2331-2390. Some preferred, butnon-limiting examples of biparatopic polypeptides of the invention aregiven in SEQ ID NO's: 2336-2390.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin); or polypeptides of the invention that comprise at least oneNanobody of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the Nanobody of the invention. Examples of polypeptides ofthe invention that comprise such half-life extending moieties or aminoacid sequences will become clear to the skilled person based on thefurther disclosure herein; and for example include, without limitation,polypeptides in which the one or more Nanobodies of the invention aresuitable linked to one or more serum proteins or fragments thereof (suchas serum albumin or suitable fragments thereof) or to one or morebinding units that can bind to serum proteins (such as, for example,Nanobodies or (single) domain antibodies that can bind to serum proteinssuch as serum albumin, serum immunoglobulins such as IgG, ortransferrine); polypeptides in which a Nanobody of the invention islinked to an Fc portion (such as a human Fc) or a suitable part orfragment thereof; or polypeptides in which the one or more Nanobodies ofthe invention are suitable linked to one or more small proteins orpeptides that can bind to serum proteins (such as, without limitation,the proteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489 and to WO 08/068,280 of Ablynx N.V.

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional Nanobodies(i.e. not directed against HER2), so as to provide a tri- ormultispecific Nanobody construct. Some preferred, but non-limitingexamples of bispecific polypeptides of the invention that bind serumalbumin are given in SEQ ID NOs: 2331-2335.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,        more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as        between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to HER2 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. In this respect, it will beclear to the skilled person that a polypeptide that contains two or moreNanobodies of the invention may bind to HER2 with an increased avidity,compared to a polypeptide that contains only one amino acid sequence ofthe invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to HER2 will become clear from the furtherdescription and examples herein.

Other polypeptides according to this preferred aspect of the inventionmay for example be chosen from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 2326-2390, in which the Nanobodies comprised within said aminoacid sequences are preferably as further defined herein.

Particularly preferred amino acid sequences of the invention (includingbut not limited to Nanobodies) and polypeptides of the invention(including polypeptides that comprise one or more Nanobodies of theinvention) are preferably such that they bind to the Herceptin® bindingsite on HER2 (and in particular domain IV of HER2, more in particularthe C-terminus of domain IV of HER2) and/or such that they can competewith Herceptin® for binding to HER-2, and also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,        more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as        between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Other particularly preferred amino acid sequences of the invention(including but not limited to Nanobodies) and polypeptides of theinvention (including polypeptides that comprise one or more Nanobodiesof the invention) are preferably such that they bind to the Omnitargbinding site on HER2 (and may in particular domain II of HER2, more inparticular the middle of domain II of HER2) and/or such that they cancompete with Omnitarg (or the Omnitarg Fab used in Example 9) forbinding to HER-2, and also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹ preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more        preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between        10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Some specifically particularly preferred polypeptides of the invention(including polypeptides that comprise one or more Nanobodies of theinvention) are preferably such that they (i) bind to the Omnitargbinding site on HER2 (and may in particular domain II of HER2, more inparticular the middle of domain II of HER2) and/or can compete withOmnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2,and (ii) bind to the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or can compete with Herceptin® for binding to HER-2, and (iii)also:

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,        more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as        between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a Nanobody of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence of theinvention (such as a Nanobody) and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid of the invention (suchas a Nanobody), at least one polypeptide of the invention and/or atleast one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with HER2. Some preferred but non-limiting applications anduses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained: (1) by isolating the V_(HH) domain of a naturally occurringheavy chain antibody; (2) by expression of a nucleotide sequenceencoding a naturally occurring V_(HH) domain; (3) by “humanization” (asdescribed herein) of a naturally occurring V_(HH) domain or byexpression of a nucleic acid encoding a such humanized V_(HH) domain;(4) by “camelization” (as described herein) of a naturally occurringV_(H) domain from any animal species, and in particular a from speciesof mammal, such as from a human being, or by expression of a nucleicacid encoding such a camelized V_(H) domain; (5) by “camelisation” of a“domain antibody” or “Dab” as described by Ward et al (supra), or byexpression of a nucleic acid encoding such a camelized V_(H) domain; (6)by using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences known per se; (7) bypreparing a nucleic acid encoding a Nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing. Suitable methods and techniques for performing theforegoing will be clear to the skilled person based on the disclosureherein and for example include the methods and techniques described inmore detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against HER2. Asfurther described herein, such V_(HH) sequences can generally begenerated or obtained by suitably immunizing a species of Camelid withHER2 (i.e. so as to raise an immune response and/or heavy chainantibodies directed against HER2), by obtaining a suitable biologicalsample from said Camelid (such as a blood sample, serum sample or sampleof B-cells), and by generating V_(HH) sequences directed against HER2,starting from said sample, using any suitable technique known per se.Such techniques will be clear to the skilled person and/or are furtherdescribed herein.

Alternatively, such naturally occurring V_(HH) domains against HER2, canbe obtained from naïve libraries of Camelid V_(HH) sequences, forexample by screening such a library using HER2, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known per se. Such libraries and techniques are forexample described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from naïve V_(HH) libraries may be used, such as V_(HH)libraries obtained from naïve V_(HH) libraries by techniques such asrandom mutagenesis and/or CDR shuffling, as for example described in WO00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against HER2. In one aspect,said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for    HER2;    and-   c) isolating the amino Nanobody or Nanobodies that can bind to    and/or have affinity for HER2.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with HER2 or with a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) Nanobodysequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made to WO03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for HER2; and (ii) cells that express heavy chain    antibodies, in which substeps (i) and (ii) can be performed    essentially as a single screening step or in any suitable order as    two separate screening steps, so as to provide at least one cell    that expresses a heavy chain antibody that can bind to and/or has    affinity for HER2;    and-   c) either (1) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with HER2 or a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone®” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against HER2 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for HER2;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withHER2 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

Also encompassed within the present invention are methods for preparingand generating multiparatopic (such as e.g. biparatopic, triparatopic,etc.) amino acids of the invention.

Without being limiting, a method for preparing and generatingbiparatopic amino acids of the invention may comprise at least the stepsof:

-   a) providing a nucleic acid sequence encoding an HER2 binding amino    acid sequence fused to a set, collection or library of nucleic acid    sequences encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for an antigenic    determinant on HER2 different from the antigenic determinant    recognized by the HER2 binding amino acid sequence;    and-   c) isolating the nucleic acid sequence encoding an HER2 binding    amino acid sequence fused to the nucleic acid sequence obtained in    b), followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences andagain screened for nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for an antigenicdeterminant on HER2 different from the antigenic determinant of the HER2binding amino acid sequence and the antigenic determinant of b) in orderto obtain a triparatopic or multiparatopic amino acid sequencerespectively.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 bindingamino acid sequence fused to the set, collection or library ofnucleotide sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

According to a particularly preferred aspect, a method for preparing andgenerating biparatopic amino acids of the invention may comprise atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,    in which each nucleic acid sequence in said set, collection or    library encodes a fusion protein that comprises a first amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 that is fused    (optionally via a linker sequence) to a second amino acid sequence,    in which essentially each second amino acid sequence (or most of    these) is a different member of a set, collection or library of    different amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2;    and-   c) isolating the nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2,    obtained in b), optionally followed by expressing the encoded amino    acid sequence.

In this preferred method, the first amino acid sequence in the fusionprotein encoded by said set collection or library of nucleic acidsequences may be the same amino acid sequence for all members of theset, collection or library of nucleic acid sequences encoding the fusionprotein; or the first amino acid sequence in the fusion protein encodedby said set collection or library of nucleic acid sequences may also bea member of a set collection or library of different amino acidsequences.

Again, in such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an HER2 bindingamino acid sequence fused to the set, collection or library ofnucleotide sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) nucleotidesequences encoding amino acid sequences will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

In step b), the set, collection or library of nucleic acid sequences mayalso be screened for nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for both the firstantigenic determinant, part, domain or epitope on HER2 and the secondantigenic determinant, part, domain or epitope on HER2. This may forexample be performed in a subsequent steps (i.e. by in a first stepscreening or selecting for nucleic acid sequences that encode an aminoacid sequence that can bind to and/or has affinity for the secondantigenic determinant, part, domain or epitope on HER2, and subsequentlyin a second step selecting or screening for nucleic acid sequences thatencode an amino acid sequence that can bind to and/or has affinity forthe first antigenic determinant, part, domain or epitope on HER2; orvisa versa) or in a single step (i.e. by simultaneously screening orselecting for nucleic acid sequences that encode an amino acid sequencethat can bind to and/or has affinity for both the first antigenicdeterminant, part, domain or epitope on HER2 and the second antigenicdeterminant, part, domain or epitope on HER2).

In a preferred aspect of the above method, the first amino acid sequenceused in step a) is preferably such that (i) it can bind to and/or hasaffinity for the Herceptin® binding site on HER2 (and may in particularbe directed against domain IV of HER2, more in particular the C-terminusof domain IV of HER2) and/or (ii) competes with Herceptin® for bindingto HER-2; and in step b), the set, collection or library of nucleic acidsequences is screened for nucleic acid sequences that encode (i) anamino acid sequence that can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) an aminoacid sequence that can compete with Omnitarg (or the Omnitarg Fab usedin Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) ispreferably such that (i) it can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) competeswith Omnitarg for binding to HER-2; and in step b), the set, collectionor library of nucleic acid sequences is screened for nucleic acidsequences that encode (i) an amino acid sequence that can bind to and/orhas affinity for the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or (ii) an amino acid sequence that can compete withHerceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

It is also possible, in step b), to screen for nucleic acid sequencesthat both (i) encode an amino acid sequence that can bind to and/or hasaffinity for the Omnitarg binding site on HER2 (and in particular domainII of HER2, more in particular the middle of domain II of HER2) and/orthat can compete with Omnitarg® (or the Omnitarg Fab used in Example 9)for binding to HER-2; and that also (ii) encode an amino acid sequencethat can bind to and/or has affinity for the Herceptin® binding site onHER2 (and in particular domain IV of HER2, more in particular theC-terminus of domain IV of HER2) and/or that can compete with Herceptin®for binding to HER-2. Again, this may be performed in separate steps ora single step, and by selecting or screening in the presence ofHerceptin® and/or Omnitarg, as applicable.

It will also be clear to the skilled person that the above methods maybe performed by screening a set, collection or library of amino acidsequences that correspond to (e.g. are encoded by) the nucleic acidsequences used in the above method; and such methods form furtheraspects of the invention.

The invention in a further aspect provides a method for preparing andgenerating biparatopic amino acids of the invention which comprises atleast the steps of:

-   a) providing a set, collection or library of nucleic acid sequences,    in which each nucleic acid sequence in said set, collection or    library encodes a fusion protein that comprises a first amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 that is fused via a    linker sequence to a second amino acid sequence that has can bind to    and/or has affinity for a second antigenic determinant, part, domain    or epitope on HER2 (which may be the same or different as the first    antigenic determinant, part, domain or epitope on HER2), in which    essentially each nucleic acid sequence (or most of these) encodes a    fusion protein with a different linker sequence so as to provide a    set, collection or library encoding different fusion proteins;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for the first and    second antigenic determinant, part, domain or epitope on HER2;    and-   c) isolating the nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for the first and    second antigenic determinant, part, domain or epitope on HER2,    optionally followed by expressing the encoded amino acid sequence.

As will be clear to the skilled person, this method can be used toscreen for suitable or even optimal linker lengths for linking the firstand second amino acid sequence. For example, in this aspect, the firstamino acid sequence may be an amino acid sequence that can bind toand/or has affinity for the Omnitarg binding site on HER2 (and may inparticular domain II of HER2, more in particular the middle of domain IIof HER2) and/or that can compete with Omnitarg (or the Omnitarg Fab usedin Example 9); and the second amino acid sequence may be an amino acidsequence that can bind to and/or has affinity for the Herceptin® bindingsite on HER2 (and in particular domain IV of HER2, more in particularthe C-terminus of domain IV of HER2) and/or that can compete withHerceptin® for binding to HER-2 (or visa versa). The screening andselection step b) may be performed as further described above.

Another method for preparing and generating biparatopic amino acids ofthe invention may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for a set, collection or library of nucleic acid sequences    that encode an amino acid sequence that can bind to and/or has    affinity for HER2;-   c) ligating said set, collection or library of nucleic acid    sequences that encode an amino acid sequence that can bind to and/or    has affinity for HER2 to another nucleic acid sequence that encodes    an amino acid sequence that can bind to and/or has affinity for HER2    (e.g. a nucleic acid sequence that encodes an amino acid sequence    that competes with Herceptin® for binding HER2);    and-   d) from the set, collection or library of nucleic acid sequences    obtained in c), isolating the nucleic acid sequences encoding a    biparatopic amino acid sequence that can bind to and/or has affinity    for HER2 (and e.g. further selecting for nucleic acid sequences that    encode a biparatopic amino acid sequence that antagonizes with    higher potency compared to the monovalent amino acid sequences),    followed by expressing the encoded amino acid sequence.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences thatcan bind to and/or have affinity for HER2 in order to obtain atriparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 can beobtained by any selection or screening method known in the art for theselection and/or screening of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 andas, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on aphage, phagemid, ribosome or suitable micro-organism (such as yeast),such as to facilitate screening. Suitable methods, techniques and hostorganisms for displaying and screening (a set, collection or library of)nucleotide sequences encoding amino acid sequences will be clear to theperson skilled in the art, for example on the basis of the furtherdisclosure herein. Reference is also made to the review by Hoogenboom inNature Biotechnology, 23, 9, 1105-1116 (2005).

Another method for preparing and generating biparatopic amino acids ofthe invention may comprise at least the steps of:

-   a) providing a first set, collection or library of nucleic acid    sequences encoding amino acid sequences;-   b) screening said first set, collection or library of nucleic acid    sequences for a nucleic acid sequence that encodes an amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2;-   c) ligating the nucleic acid sequence encoding said amino acid    sequence that can bind to and/or has affinity for a first antigenic    determinant, part, domain or epitope on HER2 to another set,    collection or library of nucleic acid sequences encoding amino acid    sequences to obtain a set, collection or library of nucleic acid    sequences that encode fusion proteins;-   d) screening said set, collection or library of nucleic acid    sequences obtained in step c) for a nucleic acid sequence that    encodes an amino acid sequence that can bind a second antigenic    determinant, part, domain or epitope on HER2 different from the    first antigenic determinant, part, domain or epitope on HER-2;    and    -   e) isolating the nucleic acid sequence that encodes an amino        acid sequence that can bind to and/or has affinity for the first        and second antigenic determinant, part, domain or epitope on        HER2, optionally followed by expressing the encoded amino acid        sequence.

In a preferred aspect of the above method, the first amino acid sequenceused in step a) is preferably such that (i) it can bind to and/or hasaffinity for the Herceptin® binding site on HER2 (and may in particularbe directed against domain IV of HER2, more in particular the C-terminusof domain IV of HER2) and/or (ii) competes with Herceptin® for bindingto HER-2; and in step b), the set, collection or library of nucleic acidsequences is screened for nucleic acid sequences that encode (i) anamino acid sequence that can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) an aminoacid sequence that can compete with Omnitarg (or the Omnitarg Fab usedin Example 9) for binding to HER-2.

Alternatively, the first amino acid sequence used in step a) ispreferably such that (i) it can bind to and/or has affinity for theOmnitarg binding site on HER2 (and may in particular domain II of HER2,more in particular the middle of domain II of HER2) and/or (ii) competeswith Omnitarg for binding to HER-2; and in step b), the set, collectionor library of nucleic acid sequences is screened for nucleic acidsequences that encode (i) an amino acid sequence that can bind to and/orhas affinity for the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or (ii) an amino acid sequence that can compete withHerceptin® for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

It is also possible, in step b), to screen for nucleic acid sequencesthat both (i) encode an amino acid sequence that can bind to and/or hasaffinity for the Omnitarg binding site on HER2 (and in particular domainII of HER2, more in particular the middle of domain II of HER2) and/orthat can compete with Omnitarg (or the Omnitarg Fab used in Example 9)for binding to HER-2; and that also (ii) encode an amino acid sequencethat can bind to and/or has affinity for the Herceptin® binding site onHER2 (and in particular domain IV of HER2, more in particular theC-terminus of domain IV of HER2) and/or that can compete with Herceptin®for binding to HER-2. Again, this may be performed in separate steps ora single step, and by selecting or screening in the presence ofHerceptin® and/or Omnitarg, as applicable.

The biparatopic amino acid sequence obtained in the method above, cansubsequently be fused to one or more further sets, collections orlibraries of nucleic acid sequences encoding amino acid sequences thatcan bind to and/or have affinity for HER2 in order to obtain atriparatopic or multiparatopic amino acid sequence respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

The set, collection or library of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 can beobtained by any selection or screening method known in the art for theselection and/or screening of nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for HER2 andas, for example, described in the Examples section.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER2 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence may be displayed on aphage, phagemid, ribosome or suitable micro-organism (such as yeast),such as to facilitate screening. Suitable methods, techniques and hostorganisms for displaying and screening (a set, collection or library of)nucleotide sequences encoding amino acid sequences will be clear to theperson skilled in the art, for example on the basis of the furtherdisclosure herein. Reference is also made to the review by Hoogenboom inNature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al. (Nat Biotechnol 23:1105,2005) and Binz et al. (Nat Biotechnol 2005, 23:1247)). Such set,collection or library of sequences can be displayed on the surface of aphage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell,and linked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against HER2 involves suitably immunizing atransgenic mammal that is capable of expressing heavy chain antibodies(i.e. so as to raise an immune response and/or heavy chain antibodiesdirected against HER2), obtaining a suitable biological sample from saidtransgenic mammal that contains (nucleic acid sequences encoding) saidV_(HH) sequences or Nanobody sequences (such as a blood sample, serumsample or sample of B-cells), and then generating V_(HH) sequencesdirected against HER2, starting from said sample, using any suitabletechnique known per se (such as any of the methods described herein or ahybridoma technique). For example, for this purpose, the heavy chainantibody-expressing mice and the further methods and techniquesdescribed in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens etal. (Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5) can beused. For example, such heavy chain antibody expressing mice can expressheavy chain antibodies with any suitable (single) variable domain, suchas (single) variable domains from natural sources (e.g. human (single)variable domains, Camelid (single) variable domains or shark (single)variable domains), as well as for example synthetic or semi-synthetic(single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized Nanobodies of the invention can be obtained inany suitable mariner known per se (i.e. as indicated under points(1)-(8) above) and thus are not strictly limited to polypeptides thathave been obtained using a polypeptide that comprises a naturallyoccurring V_(HH) domain as a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein. Such “camelizing” substitutions arepreferably inserted at amino acid positions that form and/or are presentat the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678 and Davies andRiechmann (1994 and 1996), supra). Preferably, the V₁ sequence that isused as a starting material or starting point for generating ordesigning the camelized Nanobody is preferably a V_(H) sequence from amammal, more preferably the V_(H) sequence of a human being, such as aV_(H)3 sequence. However, it should be noted that such camelizedNanobodies of the invention can be obtained in any suitable manner knownper se (i.e. as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(H) domain as astarting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” Nanobody of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired Nanobody of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for peptide synthesis known per se. Also, based on the aminoacid sequence or nucleotide sequence of a naturally occurring V_(HH)domain or V_(H) domain, respectively, a nucleotide sequence encoding thedesired humanized or camelized Nanobody of the invention, respectively,can be designed and then synthesized de novo using techniques fornucleic acid synthesis known per se, after which the nucleic acid thusobtained can be expressed in a manner known per se, so as to provide thedesired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example comprise combiningone or more parts of one or more naturally occurring V₁₄ sequences (suchas one or more FR sequences and/or CDR sequences), one or more parts ofone or more naturally occurring V_(HH) sequences (such as one or more FRsequences or CDR sequences), and/or one or more synthetic orsemi-synthetic sequences, in a suitable manner, so as to provide aNanobody of the invention or a nucleotide sequence or nucleic acidencoding the same (which may then be suitably expressed). Nucleotidesequences encoding framework sequences of V_(HH) sequences or Nanobodieswill be clear to the skilled person based on the disclosure hereinand/or the further prior art cited herein (and/or may alternatively beobtained by PCR starting from the nucleotide sequences obtained usingthe methods described herein) and may be suitably combined withnucleotide sequences that encode the desired CDR's (for example, by PCRassembly using overlapping primers), so as to provide a nucleic acidencoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized bythe presence of one or more “Hallmark residues” (as described herein) inone or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody against HER2 according to the invention mayhave the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:

-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:

-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which

-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:

-   d) CDR', CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:

-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:

-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:

-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:

-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:

-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:

-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as    described herein) and the amino acid residue at position 108 is Q;    or in which:-   ii) the amino acid residues at positions 43-46 according to the    Kabat numbering form the sequence KERE or KQRE (or a KERE-like    sequence as described) and the amino acid residue at position 108 is    Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table A-3 below. More generally, and    without limitation, Nanobodies belonging to the GLEW-group can be    defined as Nanobodies with a G at position 44 and/or with a W at    position 47, in which position 46 is usually E and in which    preferably position 45 is not a charged amino acid residue and not    cysteine;-   ii) The “KERE-group”: Nanobodies with the amino acid sequence KERE    or KQRE (or another KERE-like sequence) at positions 43-46 according    to the Kabat numbering and Q or L at position 108 according to the    Kabat numbering. As further described herein, Nanobodies within this    group usually have a F at position 37, an L or F at position 47; and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. More generally, and without limitation, Nanobodies    belonging to the KERE-group can be defined as Nanobodies with a K, Q    or R at position 44 (usually K) in which position 45 is a charged    amino acid residue or cysteine, and position 47 is as further    defined herein;-   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW at positions 44-47 according to the Kabat numbering or    the amino acid sequence KERE or KQRE at positions 43-46 according to    the Kabat numbering, the latter most preferably in combination with    an F at position 37 and an L or an F at position 47 (as defined for    the KERE-group); and can have Q or L at position 108 according to    the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics of) two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P, R,S residues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V_(H) domain would form(part of) the V_(H)/V_(L), interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2). Such substitutions include, butare not limited to, the GLEW-like sequences mentioned in Table A-3below; as well as the substitutions that are described in theInternational Application WO 00/29004 for so-called “microbodies”, e.g.so as to obtain a Nanobody with Q at position 108 in combination withKLEW at positions 44-47. Other possible substitutions at these positionswill be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of R, K, N,E, G, I, T and Q; and is most preferably either K or E (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described herein). The amino acid residue atposition 84 is chosen from the group consisting of P, A, R, S, D T, andV in one aspect, and is most preferably P (for Nanobodies correspondingto naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies,as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table A-3.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table A-4. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; predominantly L L, M, S, V, W; preferably L  37 V, I,F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾or E⁽³⁾  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferablyW⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Qor T; preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, A, L, R,S, T, D, V; preferably P predominantly A 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S;preferably W 104 G G or D; preferably G 108 L, M or T; Q, L⁽⁷⁾ or R;preferably Q or L⁽⁷⁾ predominantly L Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG atpositions 43-47. Alternatively, also sequences such as TERE (for exampleTEREL), KECE (for example KECEL or KECER), RERE (for example REREG),QERE (for example QEREG), KGRE (for example KGREG), KDRE (for exampleKDREV) are possible. Some other possible, but less preferred sequencesinclude for example DECKL and NVCEL. ⁽⁴⁾With both GLEW at positions44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP or EP atpositions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾In particular,but not exclusively, in combination with GLEW at positions 44-47.⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position 108 isalways Q in (non-humanized) V_(HH) sequences that also contain a W at103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences at positions44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW,GPEW, EWLP, GPER, GLER and ELEW.

TABLE A-4 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5to A-8 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1).

In Tables A-5-A-8, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 1118 V_(HH) sequences(data kindly provided by David Lutje Hulsing and Prof. Theo Verrips ofUtrecht University). The values for the V_(HH) entropy and the V_(HH)variability provide a measure for the variability and degree ofconservation of amino acid residues between the 1118 V_(HH) sequencesanalyzed: low values (i.e. <1, such as <0.5) indicate that an amino acidresidue is highly conserved between the V_(HH) sequences (i.e. littlevariability). For example, the G at position 8 and the G at position 9have values for the V_(HH) entropy of 0.1 and 0 respectively, indicatingthat these residues are highly conserved and have little variability(and in case of position 9 is G in all 1118 sequences analysed), whereasfor residues that form part of the CDR's generally values of 1.5 or moreare found (data not shown). Note that (1) the amino acid residues listedin the second column of Tables A-5-A-8 are based on a bigger sample thanthe 1118 V_(HH) sequences that were analysed for determining the V_(HH)entropy and V_(HH) variability referred to in the last two columns; and(2) the data represented below support the hypothesis that the aminoacid residues at positions 27-30 and maybe even also at positions 93 and94 already form part of the CDR's (although the invention is not limitedto any specific hypothesis or explanation, and as mentioned above,herein the numbering according to Kabat is used). For a generalexplanation of sequence entropy, sequence variability and themethodology for determining the same, see Oliveira et al., PROTEINS:Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q Q, A, E — — 2V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D,Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V,A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 515 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 322 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 625 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R,L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F,V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37Hallmark residue: F⁽¹⁾, H, I, L, 1.1 6 Y or V, preferably F⁽¹⁾ or Y 38 RR 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, TP, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, 1.3 5 R, S, L; preferably G⁽²⁾,E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾ 45 Hallmark residue: L⁽²⁾, R⁽³⁾,C, I, L, 0.6 4 P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 46 E, V E, D, K, Q, V0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or 1.9 9 F⁽¹⁾, A, G, I, M, R, S, Vor Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A, G A,S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F,L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.54 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R,S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F,G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 QQ, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N, G N, D, G, H, S, T0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V 0.1 2 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, 0.9 7 Q or T; preferably K or R;most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; 0.7 6preferably P 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 388 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 191 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H,K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, P⁽⁶⁾, 0.4 2 R⁽⁶⁾, S; preferably W 104 Hallmark residue: Gor D; 0.1 1 preferably G 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or 0.4 3 R; preferably Qor L⁽⁷⁾ 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an amino acid sequence with the (general)structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-3;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which. CDR1 to CDR3 refer to the complementarity determining regions1 to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    A-3 (it being understood that V_(HH) sequences will contain one or    more Hallmark residues; and that partially humanized Nanobodies will    usually, and preferably, [still] contain one or more Hallmark    residues [although it is also within the scope of the invention to    provide—where suitable in accordance with the invention—partially    humanized Nanobodies in which all Hallmark residues, but not one or    more of the other amino acid residues, have been humanized]; and    that in fully humanized Nanobodies, where suitable in accordance    with the invention, all amino acid residues at the positions of the    Hallmark residues will be amino acid residues that occur in a human    V_(H)3 sequence. As will be clear to the skilled person based on the    disclosure herein that such V_(HH) sequences, such partially    humanized Nanobodies with at least one Hallmark residue, such    partially humanized Nanobodies without Hallmark residues and such    fully humanized Nanobodies all form aspects of this invention);    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded;    and in which:-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

TABLE A-9 Representative amino acid sequences for Nanobodies of theKERE, GLEW and P, R, S 1.03 group. The CDR's are indicated with XXXXKERE sequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ ID NO: 2QVKLEESGGGLVQAGGSLRLSCVGSGRTESXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ ID NO: 3AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWERQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ IDNO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ IDNO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ IDNO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ IDNO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ IDNO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ IDNO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ IDNO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ IDNO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ IDNO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ IDNO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ IDNO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTSRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ IDNO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS

In particular, a Nanobody of the invention of the KERE group can be anamino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:

-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-10 Representative FW1 sequences for Nanobodies of theKERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of theKERE-group. KERE FW2 SEQ ID NO: 41 WFRQAPGKEREFVA sequence no. 1 KEREFW2 SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2 KERE FW2 SEQ ID NO: 43WYRQAPGKQREMVA sequence no. 3 KERE FW2 SEQ ID NO: 44 WYRQGPGKQRELVAsequence no. 4 KERE FW2 SEQ ID NO: 45 WIRQAPGKEREGVS sequence no. 5 KEREFW2 SEQ ID NO: 46 WFREAPGKEREGIS sequence no. 6 KERE FW2 SEQ ID NO: 47WYRQAPGKERDLVA sequence no. 7 KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVSsequence no. 8 KERE FW2 SEQ ID NO: 49 WFRQPPGKVREFVG sequence no. 9and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-12 Representative FW3 sequences for Nanobodies of theKERE-group. KERE FW3 SEQ ID NO: 50 RFTISRDNAKNTVYLQMNSL sequence no. 1KPEDTAVYRCYF KERE FW3 SEQ ID NO: 51 RFAISRDNNKNTGYLQMNSL sequence no. 2EPEDTAVYYCAA KERE FW3 SEQ ID NO: 52 RFTVARNNAKNTVNLEMNSL sequence no. 3KPEDTAVYYCAA KERE FW3 SEQ ID NO: 53 RFTISRDIAKNTVDLLMNNL sequence no. 4EPEDTAVYYCAA KERE FW3 SEQ ID NO: 54 RLTISRDNAVDTMYLQMNSL sequence no. 5KPEDTAVYYCAA KERE FW3 SEQ ID NO: 55 RFTISRDNAKNTVYLQMDNV sequence no. 6KPEDTAIYYCAA KERE FW3 SEQ ID NO: 56 RFTISKDSGKNTVYLQMTSL sequence no. 7KPEDTAVYYCAT KERE FW3 SEQ ID NO: 57 RFTISRDSAKNMMYLQMNNL sequence no. 8KPQDTAVYYCAA KERE FW3 SEQ ID NO: 58 RFTISRENDKSTVYLQLNSL sequence no. 9KPEDTAVYYCAA KERE FW3 SEQ ID NO: 59 RFTISRDYAGNTAYLQMNSL sequence no. 10KPEDTGVYYCATand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences for Nanobodies of theKERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ IDNO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four amino acid sequences (i.e.amino acid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables A-5 to A-8) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. KERE FW1 SEQ ID NO: 32VESGGGLVQPGGSLRLSCAASG sequence no. 10 KERE FW1 SEQ ID NO: 33VDSGGGLVQAGDSLKLSCALTG sequence no. 11 KERE FW1 SEQ ID NO: 34VDSGGGLVQAGDSLRLSCAASG sequence no. 12 KERE FW1 SEQ ID NO: 35VDSGGGLVEAGGSLRLSCQVSE sequence no. 13 KERE FW1 SEQ ID NO: 36QDSGGGSVQAGGSLKLSCAASG sequence no. 14 KERE FW1 SEQ ID NO: 37VQSGGRLVQAGDSLRLSCAASE sequence no. 15 KERE FW1 SEQ ID NO: 38VESGGTLVQSGDSLKLSCASST sequence no. 16 KERE FW1 SEQ ID NO: 39MESGGDSVQSGGSLTLSCVASG sequence no. 17 KERE FW1 SEQ ID NO: 40QASGGGLVQAGGSLRLSCSASV sequence no. 18and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the KERE-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-15 Representative FW1 sequences for Nanobodies of theGLEW-group. GLEW FW1 SEQ ID NO: 64 QVQLVESGGGLVQPGGSLRL sequence no. 1SCAASGFTFS GLEW FW1 SEQ ID NO: 65 EVHLVESGGGLVRPGGSLRL sequence no. 2SCAAFGFIFK GLEW FW1 SEQ ID NO: 66 QVKLEESGGGLAQPGGSLRL sequence no. 3SCVASGFTFS GLEW FW1 SEQ ID NO: 67 EVQLVESGGGLVQPGGSLRL sequence no. 4SCVCVSSGCT GLEW FW1 SEQ ID NO: 68 EVQLVESGGGLALPGGSLTL sequence no. 5SCVFSGSTFSand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for Nanobodies of theGLEW-group. GLEW FW2 SEQ ID NO: 72 WVRQAPGKVLEWVS sequence no. 1 GLEWFW2 SEQ ID NO: 73 WVRRPPGKGLEWVS sequence no. 2 GLEW FW2 SEQ ID NO: 74WVRQAPGMGLEWVS sequence no. 3 GLEW FW2 SEQ ID NO: 75 WVRQAPGKEPEWVSsequence no. 4 GLEW FW2 SEQ ID NO: 76 WVRQAPGKDQEWVS sequence no. 5 GLEWFW2 SEQ ID NO: 77 WVRQAPGKAEEWVS sequence no. 6 GLEW FW2 SEQ ID NO: 78WVRQAPGKGLEWVA sequence no. 7 GLEW FW2 SEQ ID NO: 79 WVRQAPGRATEWVSsequence no. 8and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-17 Representative FW3 sequences for Nanobodies of theGLEW-group. GLEW FW3 SEQ ID NO: 80 RFTISRDNAKNTLYLQMNSLK sequence no. 1PEDTAVYYCVK GLEW FW3 SEQ ID NO: 81 RFTISRDNARNTLYLQMDSLIP sequence no. 2EDTALYYCAR GLEW FW3 SEQ ID NO: 82 RFTSSRDNAKSTLYLQMNDLK sequence no. 3PEDTALYYCAR GLEW FW3 SEQ ID NO: 83 RFIISRDNAKNTLYLQMNSLGP sequence no. 4EDTAMYYCQR GLEW FW3 SEQ ID NO: 84 RFTASRDNAKNTLYLQMNSLKS sequence no. 5EDTARYYCAR GLEW FW3 SEQ ID NO: 85 RFTISRDNAKNTLYLQMDDLQS sequence no. 6EDTAMYYCGRand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences for Nanobodies of theGLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ IDNO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSSGLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no.6 SEQ ID NO: 91 WGQGTQVTVSSand in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. GLEW FW1 SEQ ID NO: 69VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW FW1 SEQ ID NO: 70EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1 SEQ ID NO: 71VESGGGLALPGGSLTLSCVFSG sequence no. 8and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the GLEW-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-20 Representative FW1 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW1 SEQ ID NO: 92 AVQLVESGGGLVQAGGSLRL sequenceno. 1 SCAASGRTFS P, R, S 103 FW1 SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLsequence no. 2 SCAASGFDFG P, R, S 103 FW1 SEQ ID NO: 94EVHLVESGGGLVRPGGSLRL sequence no. 3 SCAAFGFIFK P, R, S 103 FW1 SEQ IDNO: 95 QVQLAESGGGLVQPGGSLKL sequence no. 4 SCAASRTIVS P, R, S 103 FW1SEQ ID NO: 96 QEHLVESGGGLVDIGGSLRL sequence no. 5 SCAASERIFS P, R, S 103FW1 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRL sequence no. 6 SCVASGFTFS P, R, S103 FW1 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRL sequence no. 7 SCVCVSSGCT P,R, S 103 FW1 SEQ ID NO: 99 EVQLVESGGGLALPGGSLTL sequence no. 8SCVFSGSTFSand in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW2 SEQ ID NO: 102 WFRQAPGKEREFVA sequence no. 1P, R, S 103 FW2 SEQ ID NO: 103 WVRQAPGKVLEWVS sequence no. 2 P, R, S 103FW2 SEQ ID NO: 104 WVRRPPGKGLEWVS sequence no. 3 P, R, S 103 FW2 SEQ IDNO: 105 WIRQAPGKEREGVS sequence no. 4 P, R, S 103 FW2 SEQ ID NO: 106WVRQYPGKEPEWVS sequence no. 5 P, R, S 103 FW2 SEQ ID NO: 107WFRQPPGKEHEFVA sequence no. 6 P, R, S 103 FW2 SEQ ID NO: 108WYRQAPGKRTELVA sequence no. 7 P, R, S 103 FW2 SEQ ID NO: 109WLRQAPGQGLEWVS sequence no. 8 P, R, S 103 FW2 SEQ ID NO: 110WLRQTPGKGLEWVG sequence no. 9 P, R, S 103 FW2 SEQ ID NO: 111WVRQAPGKAEEFVS sequence no. 10and in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW3 SEQ ID NO: 112 RFTISRDNAKNTVYLQMNS sequenceno. 1 LKPEDTAVYYCAA P, R, S 103 FW3 SEQ ID NO: 113 RFTISRDNARNTLYLQMDSsequence no. 2 LIPEDTALYYCAR P, R, S 103 FW3 SEQ ID NO: 114RFTISRDNAKNEMYLQMNN sequence no. 3 LKTEDTGVYVVCGA P, R, S 103 FW3 SEQ IDNO: 115 RFTISSDSNRNMIYLQMNN sequence no. 4 LKPEDTAVYYCAA P, R, S 103 FW3SEQ ID NO: 116 RFTISRDNAKNMLYLHLNN sequence no. 5 LKSEDTAVYYCRR P, R, S103 FW3 SEQ ID NO: 117 RFTISRDNAKKTVYLRLNS sequence no. 6 LNPEDTAVYSCNLP, R, S 103 FW3 SEQ ID NO: 118 RFKISRDNAKKTLYLQMNS sequence no. 7LGPEDTAMYYCQR P, R, S 103 FW3 SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNSsequence no. 8 LKPEDTADYYCAVand in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S103-group. P, R, S 103 FW4 SEQ ID NO: 120 RGQGTQVTVSS sequence no. 1 P,R, S 103 FW4 SEQ ID NO: 121 LRGGTQVTVSS sequence no. 2 P, R, S 103 FW4SEQ ID NO: 122 GNKGTLVTVSS sequence no. 3 P, R, S 103 FW4 SEQ ID NO: 123SSPGTQVTVSS sequence no. 4 P, R, S 103 FW4 SEQ ID NO: 124 SSQGTLVTVSSsequence no. 5 P, R, S 103 FW4 SEQ ID NO: 125 RSRGIQVTVSS sequence no. 6and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P, R, S 103 class may be an aminoacid sequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 SEQ ID NO: 100VESGGGLVQAGGSLRLSC sequence no. 9 AASG P, R, S 103 FW1 SEQ ID NO: 101AESGGGLVQPGGSLKLSC sequence no. 10 AASRand in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    Nanobodies of the P, R, S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 2051-2325. This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said Nanobody and one or more of the sequencesof SEQ ID NO's: 2051-2325, in which the amino acid residues that formthe framework regions are disregarded. Such Nanobodies can be as furtherdescribed herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 2051-2325 or fromthe group consisting of from amino acid sequences that have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with at least one ofthe amino acid sequences of SEQ ID NO's: 2051-2325.

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding amino acid sequence of SEQ ID NO's: 2051-2325, a    conservative amino acid substitution, (as defined herein);    and/or:-   ii) its amino acid sequence preferably contains either only amino    acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding amino    acid sequence of SEQ ID NO's: 2051-2325;    and/or-   iii) the CDR's may be CDR's that are derived by means of maturation,    for example starting from the CDR's of to the corresponding amino    acid sequence of SEQ ID NO's: 2051-2325.

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to HER2 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER2 with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about        10⁷ M⁻¹ s⁻¹, preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹,        more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as        between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹;        and/or such that they:    -   bind to HER2 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind toHER2 with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 μM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at least one of the Hallmark residuesin FR2 and/or FR4 (again, including those at positions 108, 103 and/or45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's 2051-2325. Thus,according to one aspect of the invention, the term “Nanobody of theinvention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables A-5 toA-8 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables A-5 to A-8 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to HER2 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off) rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein for the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: 2051-2325.

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and Rat position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P,R,S-103group” or the “KERB group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a camelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein). This can generally be performed usingmethods and techniques known per se, which will be clear to the skilledperson, for example from the handbooks and references cited herein, thebackground art cited herein and/or from the further description herein.Alternatively, a nucleic acid encoding the desired analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can then be expressed as described herein. Yetanother technique may involve combining one or more naturally occurringand/or synthetic nucleic acid sequences each encoding a part of thedesired analog, and then expressing the combined nucleic acid sequenceas described herein. Also, the analogs can be provided using chemicalsynthesis of the pertinent amino acid sequence using techniques forpeptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNanobodies of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e. amino acidsequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables A-5-A-8. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V_(H) domain). Generally, however, such camelizingsubstitutions are preferably such that the resulting an amino acidsequence at least contains (a) a Q at position 108; and/or (b) a chargedamino acid or a cysteine residue at position 45 and preferably also an Eat position 44, and more preferably E at position 44 and R at position45; and/or (c) P, R or S at position 103; and optionally one or morefurther camelizing substitutions. More preferably, the camelizingsubstitutions are such that they result in a Nanobody of the inventionand/or in an analog thereof (as defined herein), such as in a humanizedanalog and/or preferably in an analog that is as defined in thepreceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's: 2051-2325. Thus, according to one aspect of theinvention, the term “Nanobody of the invention” in its broadest sensealso covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length Nanobody ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to HER2with an affinity (suitably measured and/or expressed as a K_(D)-value(actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rateand/or a k_(off)-rate, or alternatively as an IC₅₀ value, as furtherdescribed herein) that is as defined herein for the Nanobodies of theinvention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs:2051-2325.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.g.enzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov. 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to,fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine and fluorescent metals such as ¹⁵²Eu or others metals fromthe lanthanide series), phosphorescent labels, chemiluminescent labelsor bioluminescent labels (such as luminal, isoluminol, theromaticacridinium ester, imidazole, acridinium salts, oxalate ester, dioxetaneor GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates ormetallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I,¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metalliccations that are particularly suited for use in in vivo, in vitro or insitu diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe), as well as chromophores and enzymes (such as malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholine esterase). Other suitable labels will beclear to the skilled person, and for example include moieties that canbe detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, ETA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethylenetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair, Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to HER2 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such amino acid sequences        will be clear to the skilled person. Some non-limiting examples        are the small peptide vectors (“Pep-trans vectors”) described in        WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,        1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9,        1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131        (2001), and the membrane translocator sequence described by Zhao        et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal        amino acid sequences for intracellular targeting of antibody        fragments are for example described by Cardinale et al.,        Methods, 34, 171 (2004). Other suitable techniques for        intracellular targeting involve the expression and/or use of        so-called “intrabodies” comprising a Nanobody of the invention,        as mentioned below;    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutatione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked, to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to WO 07/112,940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028,977,WO 08/043,821, WO 08/043,822 by Ablynx N.V. mentioned herein and WO08/068,280 by Ablynx N.V.

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example WO 08/028,977); aminoacid sequences against human serum albumin that are cross-reactive withserum albumin from at least one species of mammal, and in particularwith at least one species of primate (such as, without limitation,monkeys from the genus Macaca (such as, and in particular, cynomologusmonkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta))and baboon (Papio ursinus), reference is again made to WO 2008/028977);amino acid sequences that can bind to serum albumin in a pH independentmanner (see for example WO2008/043821) and/or amino acid sequences thatare conditional binders (see for example WO2008/043822).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further amino acid sequences maycomprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, suchas from a heavy chain antibody (as described herein) and more preferablyfrom a conventional human 4-chain antibody; and/or may form (part of)and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 orIgG4), from IgE or from another human Ig such as IgA, IgD or IgM. Forexample, WO 94/04678 describes heavy chain antibodies comprising aCamelid V_(HH) domain or a humanized derivative thereof (i.e. aNanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domain have beenreplaced by human C_(H)2 and C_(H)3 domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other amino acid sequences that can besuitably linked to the Nanobodies of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra and tothe U.S. provisional application 61/005,324 entitled “Constructscomprising single variable domains and an Fc portion derived from IgE”filed on Dec. 4, 2007 (see also the International patent application byAblynx N.V. entitled “Constructs comprising single variable domains andan Fc portion derived from IgE” with the same filing date as the presentapplication) which is incorporated herein by reference.

The amino acids sequences or Nanobodies of the invention may, forexample, be linked to an Fc portion that is capable of effecting one ormore IgE-mediated immune responses and/or that is capable of binding toeither the FcεRI receptor and/or the FcεRII receptor. The amino acidsequences or Nanobodies of the invention may be linked, optionally via asuitable linker or hinge region, to one or more constant domains, inwhich the constant domains from the first polypeptide chain and thesecond polypeptide chains together form an Fc portion that is capable ofeffecting one or more IgE-mediated immune responses and/or that iscapable of binding to either the FcεRI receptor and/or the FcεRIIreceptor. In a preferred aspect, the Fc portion is capable of binding toFcεRI with an affinity (expressed as the K_(a) value) better than 10⁶M⁻¹, preferably better than 10⁸ M⁻¹, more preferably better than 10⁹M⁻¹, such as with a K_(a) value of about 10¹⁰ M⁻¹ or 10¹¹ M⁻¹. Morepreferably the Fc portion is capable of binding to FcεRI even with anaffinity (expressed as the K_(a) value) better than 10⁶ M⁻¹, preferablybetter than 10⁷ M⁻¹, such as with a K_(a) value of about 10⁸ M⁻¹. Suchan Fc portion may comprises one or more parts, fragments, amino acidstretches or domains of the Fc portion of IgE, preferably one or more ofthose parts, fragments, amino acid stretches or domains of the Fcportion of IgE that allow IgE to bind to its receptors. Preferably theFc portion at least comprises C_(ε)4 (or a suitable part of fragmentthereof), and optionally also comprises C_(ε)3 (or a suitable part offragment thereof) and/or C_(ε)2 (or a suitable part of fragmentthereof). Even more preferably, the Fc portion essentially consist ofthree constant domains, preferably all or essentially all derived fromhuman Fc portions.

Coupling of a Nanobody of the invention to an Fc portion may also leadto an increased half-life, compared to the corresponding Nanobody of theinvention. For some applications, the use of an Fc portion and/or ofconstant domains (i.e. C_(H)2 and/or C_(H)3 domains) that conferincreased half-life without any biologically significant effectorfunction may also be suitable or even preferred. Other suitableconstructs comprising one or more Nanobodies and one or more constantdomains with increased half-life in vivo will be clear to the skilledperson, and may for example comprise two Nanobodies linked to a C_(H)3domain, optionally via a linker sequence. Generally, any fusion proteinor derivatives with increased half-life will preferably have a molecularweight of more than 50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric Fcε chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic polypeptides of the invention may also belinked to Fc portions, in order to provide polypeptide constructs of thetype that is described in U.S. provisional application 61/005,331entitled “Immunoglobulin constructs” filed on Dec. 4, 2007 (see also theInternational patent application by Ablynx N.V. entitled “Immunoglobulinconstructs” with the same filing date as the present application) whichis incorporated herein by reference.

The invention, for example also relates to compounds or constructs thatcomprises an Fc portion that is linked, optionally via a suitable linkeror hinge region, to a pair of first amino acid sequences of theinvention (preferably Nanobodies of the invention), which are linked,optionally via a suitable linker, to a pair of second amino acidsequences of the invention (preferably Nanobodies of the invention),wherein:

-   -   both of the first amino acid sequences of the invention        (preferably Nanobodies of the invention) are directed against a        first epitope, antigenic determinant, part, domain or subunit on        HER2; and    -   both of the amino acid sequences of the invention (preferably        Nanobodies of the invention) are directed against a second        epitope, antigenic determinant, part, domain or subunit on HER2        which is the same or different from said first epitope,        antigenic determinant, part, domain or subunit.        Such compounds or constructs of the invention may, for example        be directed against the Herceptin® binding site on HER-2 (and in        particular against domain IV of HER2, and more in particular        against the C-terminus of domain IV of HER2) and/or are        compounds or constructs that are capable of competing with        Herceptin® for binding to HER-2; they may be directed against        the Omnitarg® binding site on HER-2 (and in particular against        domain II of HER2, and more in particular against the middle of        domain II of HER2) and/or are compounds or constructs that are        capable of competing with Omnitarg® for binding to HER-2; or        they may be directed against the Herceptin® binding site on        HER-2 (and in particular against domain IV of HER2, and more in        particular against the C-terminus of domain IV of HER2) and/or        are compounds or constructs that are capable of competing with        Herceptin® for binding to HER-2 and simultaneously be directed        against the Omnitarg® binding site on HER-2 (and in particular        against domain II of HER2, and more in particular against the        middle of domain II of HER2) and/or are compounds or constructs        that are capable of competing with Omnitarg® for binding to        HER-2.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, the “Peptrans” vectors mentioned above, the sequencesdescribed by Cardinale et al. and the amino acid sequences and antibodyfragments known per se that can be used to express or produce theNanobodies and polypeptides of the invention as so-called “intrabodies”,for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No.7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further Nanobody, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). Polypeptides of the invention that comprise two or moreNanobodies, of which at least one is a Nanobody of the invention, willalso be referred to herein as “multivalent” polypeptides of theinvention, and the Nanobodies present in such polypeptides will also bereferred to herein as being in a “multivalent format”. For example a“bivalent” polypeptide of the invention comprises two Nanobodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three Nanobodies, optionallylinked via two linker sequences; etc.; in which at least one of theNanobodies present in the polypeptide, and up to all of the Nanobodiespresent in the polypeptide, is/are a Nanobody of the invention.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different, and may be directed against thesame antigen or antigenic determinant (for example against the samepart(s) or epitope(s) or against different parts or epitopes) or mayalternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide of the invention may comprise (a) two identicalNanobodies; (b) a first Nanobody directed against a first antigenicdeterminant of a protein or antigen and a second Nanobody directedagainst the same antigenic determinant of said protein or antigen whichis different from the first Nanobody; (c) a first Nanobody directedagainst a first antigenic determinant of a protein or antigen and asecond Nanobody directed against another antigenic determinant of saidprotein or antigen; or (d) a first Nanobody directed against a firstprotein or antigen and a second Nanobody directed against a secondprotein or antigen (i.e. different from said first antigen). Similarly,a trivalent polypeptide of the invention may, for example and withoutbeing limited thereto. comprise (a) three identical Nanobodies; (b) twoidentical Nanobody against a first antigenic determinant of an antigenand a third Nanobody directed against a different antigenic determinantof the same antigen; (c) two identical Nanobody against a firstantigenic determinant of an antigen and a third Nanobody directedagainst a second antigen different from said first antigen; (d) a firstNanobody directed against a first antigenic determinant of an antigen, asecond Nanobody directed against a second antigenic determinant of saidantigen and a third Nanobody directed against a third antigenicdeterminant of the same antigen; (e) a first Nanobody directed against afirst antigenic determinant of a first antigen, a second Nanobodydirected against a second antigenic determinant of said first antigenand a third Nanobody directed against a second antigen different fromsaid first antigen; or (f) a first Nanobody directed against a firstantigen, a second Nanobody directed against a second antigen differentfrom said first antigen, and a third Nanobody directed against a thirdantigen different from said first and second antigen.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigenicdeterminant on HER2 and at least one Nanobody is directed against asecond antigenic determinant on HER2 will also be referred to as“multiparatopic” polypeptides of the invention, and the Nanobodiespresent in such polypeptides will also be referred to herein as being ina “multiparatopic format”. Thus, for example, a “biparatopic”polypeptide of the invention is a polypeptide that comprises at leastone Nanobody directed against a first antigenic determinant on HER2 andat least one further Nanobody directed against a second antigenicdeterminant on HER2, whereas a “triparatopic” polypeptide of theinvention is a polypeptide that comprises at least one Nanobody directedagainst a first antigenic determinant on HER2, at least one furtherNanobody directed against a second antigenic determinant on HER2 and atleast one further Nanobody directed against a third antigenicdeterminant on HER2; etc.

Accordingly, in its simplest form, a biparatopic polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against a first antigenicdeterminant on HER2, and a second Nanobody directed against a secondantigenic determinant on HER2, in which said first and second Nanobodymay optionally be linked via a linker sequence (as defined herein);whereas a triparatopic polypeptide of the invention in its simplest formis a trivalent polypeptide of the invention (as defined herein),comprising a first Nanobody directed against a first antigenicdeterminant on HER2, a second. Nanobody directed against a secondantigenic determinant on HER2 and a third Nanobody directed against athird antigenic determinant on HER2, in which said first, second andthird Nanobody may optionally be linked via one or more, and inparticular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multiparatopicpolypeptide of the invention may comprise at least one Nanobody againsta first antigenic determinant on HER2, and any number of Nanobodiesdirected against one or more other antigenic determinants on HER2.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against HER2) and at least one Nanobody is directed against a secondantigen (i.e. different from HER2), will also be referred to as“multispecific” polypeptides of the invention, and the Nanobodiespresent in such polypeptides will also be referred to herein as being ina “multispecific format”. Thus, for example, a “bispecific” polypeptideof the invention is a polypeptide that comprises at least one Nanobodydirected against a first antigen (i.e. HER2) and at least one furtherNanobody directed against a second antigen (i.e. different from HER2),whereas a “trispecific” polypeptide of the invention is a polypeptidethat comprises at least one Nanobody directed against a first antigen(i.e. HER2), at least one further Nanobody directed against a secondantigen (i.e. different from HER2) and at least one further Nanobodydirected against a third antigen (i.e. different from both HER2, and thesecond antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against HER2, and a secondNanobody directed against a second antigen, in which said first andsecond Nanobody may optionally be linked via a linker sequence (asdefined herein); whereas a trispecific polypeptide of the invention inits simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first Nanobody directed against HER2, asecond Nanobody directed against a second antigen and a third Nanobodydirected against a third antigen, in which said first, second and thirdNanobody may optionally be linked via one or more, and in particular oneand more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstHER2, and any number of Nanobodies directed against one or more antigensdifferent from HER2.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for HER2, oragainst the one or more other antigens), said order or arrangement isusually not critical and may be suitably chosen by the skilled person,optionally after some limited routine experiments based on thedisclosure herein. Thus, when reference is made to a specificmultivalent or multispecific polypeptide of the invention, it should benoted that this encompasses any order or arrangements of the relevantNanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the polypeptides of the invention include the Nanobodiesdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, the some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example WO 08/028,977 by AblynxN.V.); Nanobodies against human serum albumin that are cross-reactivewith serum albumin from at least one species of mammal, and inparticular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example WO 08/028,977 byAblynx N.V.); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example WO 08/043,821 by Ablynx N.V.) and/orNanobodies that are conditional binders (see for example WO 08/043,822by Ablynx N.V.).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) isparticularly preferred.

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one Nanobody of the invention and atleast one Nanobody that provides for increased half-life are given inSEQ ID NOs: 2331-2335.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding Nanobody of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent, multiparatopic andmultispecific polypeptides will be clear to the skilled person, and maygenerally be any linker or spacer used in the art to link amino acidsequences. Preferably, said linker or spacer is suitable for use inconstructing proteins or polypeptides that are intended forpharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 75, preferably between1 and 60, more preferably between 1 and 50, even more preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS35, GS30 (SEQ ID NO: 85 in WO 06/122825)and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, polyethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for HER2, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer.

Similarly, in a multiparatopic polypeptide of the invention thatcomprises Nanobodies directed against two or more different antigenicdeterminants on the same antigen (for example against different epitopesof an antigen and/or against different subunits of a multimeric receptoror protein), the length and flexibility of the linker are preferablysuch that, when the multiparatopic polypeptide binds to HER-2, at leasttwo and preferably all of the Nanobodies that are present in themultiparatopic polypeptide can (simultaneously) bind to each of theirintended antigenic determinants, epitopes, parts or domains, mostpreferably so as to allow binding with increased avidity and alsointramolecular binding and/or recognition. Again, based on thedisclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

For example, as further described herein, some of the most preferredmultiparatopic polypeptides of the invention comprise (i) at least oneamino acid sequence of the invention (and in particular at least oneNanobody) that is directed against the Omnitarg binding site on HER2(and in particular against domain II of HER2, and more in particularagainst the middle of domain II of HER2) and/or that is capable ofcompeting with Omnitarg for binding to HER-2; and at least one aminoacid sequence of the invention (and in particular at least one Nanobody)that is directed against the Herceptin® binding site on HER2 (and inparticular against domain IV of HER2, and more in particular against theC-terminus of domain IV of HER2) and/or that is capable of competingwith Herceptin® for binding to HER-2. In such a preferred multiparatopicpolypeptide of the invention, the linker is most preferably such thatthe multiparatopic polypeptide of the invention is capable of(simultaneously) binding to both the Omnitarg binding site on HER2 (andin particular against domain II of HER2, and more in particular againstthe middle of domain of HER2) as well as the Herceptin® binding site onHER2 (and in particular against domain IV of HER2, and more inparticular against the C-terminus of domain IV of HER2), again mostpreferably so as to allow binding with increased avidity and alsointramolecular binding and/or recognition. Such multiparatopicpolypeptides of the invention with such a linker form a particularlypreferred aspect of the invention, and examples of such a linker aregiven in the Examples below. For example, when such a linker is aGly-Ser linker (for example, a Gly-Ser linker as described in theExamples), it preferably has a length of at least 15 amino acidresidues, such as at least 20 or at least 30 amino acid residues. Themaximum length is not especially critical, but for practicalconsiderations (such as ease of cloning and expression) the linker ispreferably no longer than 75 amino acid residues, more preferably lessthan 50 amino acid residues. For example, Gly-Ser linkers (such as theGly-Ser linkers as described in the Examples) of between 20 and 40 aminoacid residues, such as about 25, 30 or 35 amino acid residues, may beparticularly suited. Based on the disclosure herein, the skilled personwill be able to determine other suitable linkers, it being understoodthat the optimal length of each linker may also depend on the amino acidcomposition of the linker that is envisaged for use.

Optimal linker lengths in biparatopic, triparatopic or multiparatopicpolypeptides of the invention can, for example, be designed in silicowith any method for protein design known in the art or disclosed herein(see, e.g. the Example section). Optimal linker lengths, far exampleobtained by in silico design, can further be verified experimentally bybinding and competition assays as will be known to the skilled personand/or described herein (see e.g. the example section). Optimal linkerlengths in biparatopic, triparatopic or multiparatopic polypeptides mayalso be determined using the screening method for determining optimallinker length as described herein.

The choice of linker length in biparatopic, triparatopic ormultiparatopic polypeptides of the invention can also be such that onlya limited epitope space on the antigen is covered. Linker lengthrestriction can, for example, help to avoid targeting epitopes whichshould not be neutralized (e.g. those essential for a function of theantigen) or to target regions relatively adjacent to a first ‘guiding’Nanobody.

The choice of the format (N- or C-terminal position of the differentNanobodies) of the biparatopic, triparatopic or multiparatopicpolypeptides of the invention and linker length can also be used toobtain molecules that bind avidly to the target antigen (via two, ormore, binding sites), yet are purposely not agonistic. By optimising theformat and linker length and composition, the binding sites can bepositioned in such way that simultaneous binding of two or moreNanobodies to the same target antigen (i.e. intramolecular binding) willbe highly favoured compared to binding to separate antigens in proximityof one another (intermolecular binding, such as e.g. on a cell surface).This could, for example, reduce the chance on agonism (which might notbe desired in a good therapeutic compound). Screening and/or selectionmethods and assays are known to the skilled person and/or describedherein that allow for the isolation of avidly binding domains positionedin relation to one another and to the antigen of interest in such way asto have an antagonistic function only.

In another aspect of the invention, biparatopic, triparatopic ormultiparatopic polypeptides of the invention can also be selected to bepurposely agonistic. For example, a combination of two identical or twodifferent Nanobodies that bind to the Herceptin®-binding site and aregenetically fused to one another can be agonistic (e.g. 2D3-2D3 or 2D3fused to other Herceptin®-competing Nanobodies). The current inventionalso provides a way to select for such agonistic biparatopic,triparatopic or multiparatopic polypeptides of the invention usingappropriate screening and/or selection procedures of members ofmultiparatopic libraries. Agonists could, for example, be desired and/orinteresting for triggering certain receptors.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 above) can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, Nanobodies,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, Nanobody and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of HER2 as a template. These and othertechniques will be clear to the skilled person, and reference is againmade to the standard handbooks, such as Sambrook et al. and Ausubel etal., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described herein); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used. For example, regulatory sequences, promoters and terminators    known per se for the expression and production of antibodies and    antibody fragments (including but not limited to (single) domain    antibodies and ScFv fragments) may be used in an essentially    analogous manner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in the host cells mentionedherein; and in particular promoters for the expression in the bacterialcells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycin or ampicillin), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell. For example, leader sequences known per se for theexpression and production of antibodies and antibody fragments(including but not limited to single domain antibodies and ScFvfragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those that can be used for theexpression in the host cells mentioned herein; and in particular thosethat are suitable for expression in bacterial cells, such as thosementioned herein and/or those used in the Examples below. For some(further) non-limiting examples of the promoters, selection markers,leader sequences, expression markers and further elements that may bepresent/used in the genetic constructs of the invention—such asterminators, transcriptional and/or translational enhancers and/orintegration factors—reference is made to the general handbooks such asSambrook et al. and Ausubel et al. mentioned above, as well as to theexamples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO95/21191, WO 97/11094, WO 97/42320. WO 98/06737, WO 98/21355, U.S. Pat.No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other exampleswill be clear to the skilled person. Reference is also made to thegeneral background art cited above and the further references citedherein.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy). For this purpose, thenucleotide sequences of the invention may be introduced into the cellsor tissues in any suitable way, for example as such (e.g. usingliposomes) or after they have been inserted into a suitable gene therapyvector (for example derived from retroviruses such as adenovirus, orparvoviruses such as adeno-associated virus). As will also be clear tothe skilled person, such gene therapy may be performed in vivo and/or insitu in the body of a patient by administering a nucleic acid of theinvention or a suitable gene therapy vector encoding the same to thepatient or to specific cells or a specific tissue or organ of thepatient; or suitable cells (often taken from the body of the patient tobe treated, such as explanted lymphocytes, bone marrow aspirates ortissue biopsies) may be treated in vitro with a nucleotide sequence ofthe invention and then be suitably (re-)introduced into the body of thepatient. All this can be performed using gene therapy vectors,techniques and delivery systems which are well known to the skilledperson, and for example described in Culver, K. W., “Gene Therapy”,1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.);Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, GeneTher. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.; 811 (1997),289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No.5,580,859; U.S. Pat. No. 5,589,546; or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640. For example, in situ expression of ScFvfragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and ofdiabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No.6,849,992 for general techniques for introducing transgenes intomammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or turbers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian, cell lines, in particular Chinese hamsterovary (CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, Nanobody or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, Nanobodies and the polypeptides of the invention, the aminoacid sequences, Nanobodies and polypeptides of the invention can beproduced either intracellularly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates the further isolation and downstream processingof the Nanobodies and proteins obtained. Bacterial cells such as thestrains of E. coli mentioned above normally do not secrete proteinsextracellularly, except for a few classes of proteins such as toxins andhemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmicspace. Periplasmic production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to the natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasm than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasm. Anotheradvantage is that correct disulfide bonds may form because the periplasmprovides a more oxidative environment than the cytoplasm. Proteinsoverexpressed in E. coli are often found in insoluble aggregates,so-called inclusion bodies. These inclusion bodies may be located in thecytosol or in the periplasm; the recovery of biologically activeproteins from these inclusion bodies requires a denaturation/refoldingprocess. Many recombinant proteins, including therapeutic proteins, arerecovered from inclusion bodies. Alternatively, as will be clear to theskilled person, recombinant strains of bacteria that have beengenetically modified so as to secrete a desired protein, and inparticular an amino acid sequence, Nanobody or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been produced intracellularlyand that has been isolated from the host cell, and in particular from abacterial cell or from an inclusion body in a bacterial cell. Accordingto another non-limiting aspect of the invention, the amino acidsequence, Nanobody or polypeptide of the invention is an amino acidsequence, Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left-(PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence; for        expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP 1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        ³⁵S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I);    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSGS (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterial cells: pET vectors (Novagen)        and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal;    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (pho1), invertase (Suc), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865, WO 04/041867 and WO 08/020,079) as well as tothe standard handbooks, such as Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990), Remington, the Scienceand Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins(2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.),Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andaqueous buffers and solutions such as physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution;water oils; glycerol; ethanol; glycols such as propylene glycol or aswell as mineral oils, animal oils and vegetable oils, for example peanutoil, soybean oil, as well as suitable mixtures thereof. Usually, aqueoussolutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences. Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the amino acid sequences, Nanobodies and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the amino acid sequences, Nanobodies andpolypeptides of the invention or their salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all eases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid sequences, Nanobodies and polypeptides of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the amino acid sequences, Nanobodies and polypeptidesof the invention can be dissolved or dispersed at effective levels,optionally with the aid of non-toxic surfactants. Adjuvants such asfragrances and additional antimicrobial agents can be added to optimizethe properties for a given use. The resultant liquid compositions can beapplied from absorbent pads, used to impregnate bandages and otherdressings, or sprayed onto the affected area using pump-type or aerosolsprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the amino acid sequences, Nanobodies and polypeptides of theinvention to the skin are known to the art; for example, see Jacquet etal. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith etal. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptidesof the invention can be determined by comparing their in vitro activity,and in vivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4). Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one cancer and/or tumor, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with HER2, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signalling in which HER2 is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a Nanobody of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, the invention relates toa method for the prevention and/or treatment of at least one disease ordisorder that can be treated by modulating HER2, its biological orpharmacological activity, and/or the biological pathways or signallingin which HER2 is involved, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of an aminoacid sequence of the invention, of a Nanobody of the invention, of apolypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same. In particular, said pharmaceutically effectiveamount may be an amount that is sufficient to modulate HER2, itsbiological or pharmacological activity, and/or the biological pathwaysor signalling in which HER2 is involved; and/or an amount that providesa level of the amino acid sequence of the invention, of a Nanobody ofthe invention, of a polypeptide of the invention in the circulation thatis sufficient to modulate HER2, its biological or pharmacologicalactivity, and/or the biological pathways or signalling in which HER2 isinvolved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a Nanobody of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,Nanobody or polypeptide of the invention to be used, the specific routeof administration and pharmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, Nanobodies and polypeptidesof the invention will generally be administered in an amount between 1grain and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences.Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone cancer and/or tumor; and/or for use in one or more of the methods oftreatment mentioned herein.

In another aspect, the invention relates to an amino acid sequence,Nanobody or polypeptide of the invention for prevention and/or treatmentof at least one cancer and/or tumor; and/or for use in one or more ofthe methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofcancers and/or tumors, and in particular for the prevention andtreatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other amino acid sequences and in particular (single) domainantibodies against HER2, as well as polypeptides comprising such(single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., ProteinScience (2004), 13:1882-1891; Ewert et al., Methods, 2004 October;34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7):773-783; O'Brien and Jones, Methods Mol. Biol. 2003:207:81-100; Skerra,J. Mol. Recognit. 2000:13:167-187, and Saerens et al., J. Mol. Biol.2005 Sep. 23; 352(3):597-607, and the further references cited therein.For example, techniques known per se for grafting mouse or rat CDR'sonto human frameworks and scaffolds can be used in an analogous mannerto provide chimeric proteins comprising one or more of the CDR's of theNanobodies of the invention and one or more human framework regions orsequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example from Nanobodies (preferred), V_(H) domains fromconventional antibodies (and in particular from human antibodies), heavychain antibodies, conventional 4-chain antibodies (such as conventionalhuman 4-chain antibodies) or other immunoglobulin sequences directedagainst HER2. Such immunoglobulin sequences directed against HER2 can begenerated in any manner known per se, as will be clear to the skilledperson, i.e. by immunization with HER2 or by screening a suitablelibrary of immunoglobulin sequences with HER2, or any suitablecombination thereof. Optionally, this may be followed by techniques suchas random or site-directed mutagenesis and/or other techniques foraffinity maturation known per se. Suitable techniques for generatingsuch immunoglobulin sequences will be clear to the skilled person, andfor example include the screening techniques reviewed by Hoogenboom,Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques forgenerating immunoglobulins against a specified target include forexample the Nanoclone technology (as for example described in thepublished US patent application 2006-0211088), so-called SLAM technology(as for example described in the European patent application 0 542 810),the use of transgenic mice expressing human immunoglobulins or thewell-known hybridoma techniques (see for example Larrick et al,Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be usedto generate immunoglobulins against HER2, and the CDR's of suchimmunoglobulins can be used in the Nanobodies of the invention, i.e. asoutlined above. For example, the sequence of such a CDR can bedetermined, synthesized and/or isolated, and inserted into the sequenceof a Nanobody of the invention (e.g. so as to replace the correspondingnative CDR), all using techniques known per se such as those describedherein, or Nanobodies of the invention containing such CDR's (or nucleicacids encoding the same) can be synthesized de novo, again using thetechniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifyHER2 from compositions and preparations comprising the same. Derivativesof the amino acid sequences of the invention that comprise a suitabledetectable label can also be used as markers to determine (qualitativelyor quantitatively) the presence of HER2 in a composition or preparationor as a marker to selectively detect the presence of HER2 on the surfaceof a cell or tissue (for example, in combination with suitable cellsorting techniques).

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures:

Preferred Aspects

-   1. Amino acid sequence that is directed against and/or that can    specifically bind to HER2.-   2. Amino acid sequence according to aspect 1, wherein said amino    acid sequence competes with Herceptin® for binding to HER2.-   3. Amino acid sequence according to any of aspects 1 or 2, wherein    said amino acid sequence inhibits and/or blocks binding of    Herceptin® to HER-   4. Amino acid sequence according to any of aspects 1 to 3, wherein    said amino acid sequence is directed against the Herceptin® binding    site on HER2.-   5. Amino acid sequence according to any of aspects 1 to 4, wherein    said amino acid sequence specifically binds to domain IV of HER2.-   6. Amino acid sequence according to aspect 1, wherein said amino    acid sequence competes with Omnitarg for binding to HER2.-   7. Amino acid sequence according to any of aspect 1 or 7, wherein    said amino acid sequence inhibits and/or blocks binding of Omnitarg    to HER2.-   8. Amino acid sequence according to any of aspects 6 or 7, wherein    said amino acid sequence is directed against the Omnitarg binding    site on HER2.-   9. Amino acid sequence according to any of aspects 6 to 8, wherein    said amino acid sequence specifically binds to domain II of HER2.-   10. Amino acid sequence according to any of aspects 1 to 9, wherein    said amino acid competes with Herceptin® and Omnitarg for binding to    HER2.-   11. Amino acid sequence according to any of aspects 1 to 10, wherein    said amino acid sequence inhibits and/or blocks binding of    Herceptin® and Omnitarg to HER2.-   12. Amino acid sequence according to any of aspects 1 to 11, wherein    said amino acid sequence inhibits and/or blocks tumor cell    proliferation.-   13. Amino acid sequence according to any of aspects 1 to 12, wherein    said amino acid sequence inhibits, downregulates and/or blocks cell    signalling.-   14. Amino acid sequence according to any of aspects 1 to 13, wherein    said amino acid sequence induces apoptosis in tumor cells.-   15. Amino acid sequence according to any of aspects 1 to 14, wherein    said amino acid sequence inhibits and/or blocks heterodimerization    between ERBB receptors.-   16. Amino acid sequence according to any of aspects 1 to 15, wherein    said amino acid sequence inhibits and/or blocks ligand activation of    an ERbB hetero-oligomer comprising HER2 and HER3, HER4 or EGFR.-   17. Amino acid sequence according to any of aspects 1 to 16, wherein    said amino acid sequence inhibits and/or blocks tumor    vascularisation.-   18. Amino acid sequence according to any of aspects Ito 17, wherein    said amino acid sequence recruits immune effector cells such as    macrophages and monocytes to the tumor.-   19. Amino acid sequence according to any of aspects 1 to 18, wherein    said amino acid sequence inhibits and/or blocks TNF induced    signalling and/or cell proliferation.-   20. Amino acid sequence according to any of aspects 1 to 19, wherein    said amino acid sequence downregulates HER2 levels and/or    downregulates HER2-mediated signalling pathways.-   21. Amino acid sequence according to any of aspects 1 to 20, wherein    said amino acid sequence inhibits and/or blocks    metalloproteinase-mediated HER2 ectodomain shedding.-   22. Amino acid sequence according to any of aspects 1 to 21, wherein    said amino acid sequence inhibits, downregulates and/or blocks    ligand-mediated ErbB signalling.-   23. Amino acid sequence according to any of aspects 1 to 22, wherein    said amino acid sequence inhibits and/or blocks HER2 ectodomain    cleavage.-   24. Amino acid sequence according to any of aspects 1 to 23, wherein    said amino acid sequence inhibits and/or blocks Heregulin-mediated    activation of MAPK/Erk1/2.-   25. Amino acid sequence according to any of aspects 1 to 24, wherein    said amino acid sequence inhibits and/or blocks P13K/Akt signalling.-   26. Amino acid sequence according to any of aspects 1 to 25, wherein    said amino acid sequence modulates HER2 or HER2 mediated signalling    via the same mechanism of action as Herceptin®.-   27. Amino acid sequence according to any of aspects 1 to 26, wherein    said amino acid sequence modulates HER2 or HER2 mediated signalling    via the same mechanism of action as Omnitarg.-   28. Amino acid sequence according to any of aspects 1 to 27, that is    in essentially isolated form.-   29. Amino acid sequence according to aspect 1 or 28, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.-   30. Amino acid sequence according to any of aspects 1 to 29, that    can specifically bind to HER2 with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   31. Amino acid sequence according to any of aspects 1 to 30, that    can specifically bind to HER2 with a rate of association    (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹ preferably    between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹    s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹.-   32. Amino acid sequence according to any of aspects 1 to 31, that    can specifically bind to HER2 with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   33. Amino acid sequence according to any of aspects 1 to 32, that    can specifically bind to HER2 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 μM.-   34. Amino acid sequence according to any of aspects 1 to 33, that is    a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.-   35. Amino acid sequence according to any of aspects 1 to 34, that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.-   36. Amino acid sequence according to any of aspects 1 to 35, that    essentially consists of 4 framework regions (FR1 to FR4    respectively) and 3 complementarity determining regions (CDR1 to    CDR3 respectively).-   37. Amino acid sequence according to any of aspects 1 to 36, that is    an immunoglobulin sequence.-   38. Amino acid sequence according to any of aspects 1 to 37, that is    a naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.-   39. Amino acid sequence according to any of aspects 1 to 38, that is    a humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.-   40. Amino acid sequence according to any of aspects 1 to 39, that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).-   41. Amino acid sequence according to any of aspects 1 to 40, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   42. Amino acid sequence according to any of aspects 1 to 41, that    essentially consists of a domain antibody (or an amino acid sequence    that is suitable for use as a domain antibody), of a single domain    antibody (or an amino acid sequence that is suitable for use as a    single domain antibody), of a “dAb” (or an amino acid sequence that    is suitable for use as a dAb) or of a Nanobody® (including but not    limited to a V_(HH) sequence).-   43. Amino acid sequence according to any of aspects 1 to 42, that    essentially consists of a Nanobody®.-   44. Amino acid sequence according to any of aspects 1 to 43, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   45. Amino acid sequence according to any of aspects 1 to 44, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 2051-2325, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   46. Amino acid sequence according to any of aspects 1 to 45, that    essentially consists of a humanized Nanobody®.-   47. Amino acid sequence according to any of aspects 1 to 46, that in    addition to the at least one binding site for binding against HER2,    contains one or more further binding sites for binding against other    antigens, proteins or targets.-   48. Amino acid sequence directed against HER2, that comprises one or    more stretches of amino acid residues chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775;    -   or any suitable combination thereof.-   49. Amino acid sequence according to aspect 48, in which at least    one of said stretches of amino acid residues forms part of the    antigen binding site for binding against HER2.-   50. Amino acid sequence according to any of aspects 48 or 49, that    comprises two or more stretches of amino acid residues chosen from    the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to        a), b) or c), the second stretch of amino acid residues        corresponds to one of the amino acid sequences according to d),        e), f), g), f) or i); (ii) when the first stretch of amino acid        residues corresponds to one of the amino acid sequences        according to d), e) or f), the second stretch of amino acid        residues corresponds to one of the amino acid sequences        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the amino        acid sequences according to g), h) or i), the second stretch of        amino acid residues corresponds to one of the amino acid        sequences according to a), b), c), d), e) or f).-   51. Amino acid sequence according to aspect 50, in which the at    least two stretches of amino acid residues forms part of the antigen    binding site for binding against HER2.-   52. Amino acid sequence according to any of aspects 48 to 51, that    comprises three or more stretches of amino acid residues, in which    the first stretch of amino acid residues is chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775.-   53. Amino acid sequence according to aspect 52, in which the at    least three stretches of amino acid residues forms part of the    antigen binding site for binding against HER2.-   54. Amino acid sequence according to any of aspects 48 to 53, in    which the CDR sequences of said amino acid sequence have at least    70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the amino acid    sequences of SEQ ID NO's: 2051-2325.-   55. Amino acid sequence directed against HER2 that cross-blocks the    binding of at least one of the amino acid sequences according to any    of aspects 48 to 54 to HER2.-   56. Amino acid sequence directed against HER2 that is cross-blocked    from binding to HER2 by at least one of the amino acid sequences    according to any of aspects 48 to 54.-   57. Amino acid sequence according to any of aspects 55 or 56,    wherein the ability of said amino acid sequence to cross-block or to    be cross-blocked is detected in a Biacore assay.-   58. Amino acid sequence according to any of aspects 55 to 57,    wherein the ability of said amino acid sequence to cross-block or to    be cross-blocked is detected in an ELISA assay.-   59. Amino acid sequence according to any of aspects 58 to 58, that    is in essentially isolated form.-   60. Amino acid sequence according to any of aspects 48 to 59, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.-   61. Amino acid sequence according to any of aspects 48 to 60, that    can specifically bind to HER2 with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   62. Amino acid sequence according to any of aspects 48 to 61, that    can specifically bind to HER2 with a rate of association    (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,    preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably    between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and    10⁷ M⁻¹ s⁻¹.-   63. Amino acid sequence according to any of aspects 48 to 62, that    can specifically bind to HER2 with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   64. Amino acid sequence according to any of aspects 48 to 63, that    can specifically bind to HER2 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 μM.-   65. Amino acid sequence according to any of aspects 48 to 64, that    is a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.-   66. Amino acid sequence according to any of aspects 48 to 65, that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.-   67. Amino acid sequence according to any of aspects 48 to 66, that    is an immunoglobulin sequence.-   68. Amino acid sequence according to any of aspects 48 to 67, that    is a naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.-   69. Amino acid sequence according to any of aspects 48 to 68, that    is a humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.-   70. Amino acid sequence according to any of aspects 48 to 69, that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).-   71. Amino acid sequence according to any of aspects 48 to 70, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   72. Amino acid sequence according to any of aspects 48 to 71, that    essentially consists of a domain antibody (or an amino acid sequence    that is suitable for use as a domain antibody), of a single domain    antibody (or an amino acid sequence that is suitable for use as a    single domain antibody), of a “dAb” (or an amino acid sequence that    is suitable for use as a dAb) or of a Nanobody® (including but not    limited to a V_(HH) sequence).-   73. Amino acid sequence according to any of aspects 48 to 72, that    essentially consists of a Nanobody®.-   74. Amino acid sequence according to any of aspects 48 to 73, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   75. Amino acid sequence according to any of aspects 48 to 74, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 2051-2325, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   76. Amino acid sequence according to any of aspects 48 to 75, that    essentially consists of a humanized Nanobody®.-   77. Amino acid sequence according to any of aspects 1 to 76, that in    addition to the at least one binding site for binding formed by the    CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.-   78. Amino acid sequence that essentially consists of 4 framework    regions (FR1 to FR4, respectively) and 3 complementarity determining    regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775.-   79. Amino acid sequence that essentially consists of 4 framework    regions (FR1 to FR4, respectively) and 3 complementarity determining    regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775].-   80. Amino acid sequence according to any of aspects 78 to 79, in    which the CDR sequences of said amino acid sequence have at least    70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the amino acid    sequences of SEQ ID NO's: 2051-2325.-   81. Amino acid sequence directed against HER2 that cross-blocks the    binding of at least one of the amino acid sequences according to any    of aspects 78 to 80 to HER2.-   82. Amino acid sequence directed against HER2 that is cross-blocked    from binding to HER2 by at least one of the amino acid sequences    according to any of aspects 78 to 80.-   83. Amino acid sequence according to any of aspects 81 or 82,    wherein the ability of said amino acid sequence to cross-block or to    be cross-blocked is detected in a Biacore assay.-   84. Amino acid sequence according to any of aspects 81 to 83,    wherein the ability of said amino acid sequence to cross-block or to    be cross-blocked is detected in an ELISA assay.-   85. Amino acid sequence according to any of aspects 78 to 84, that    is in essentially isolated form.-   86. Amino acid sequence according to any of aspects 78 to 85, for    administration to a subject, wherein said amino acid sequence does    not naturally occur in said subject.-   87. Amino acid sequence according to any of aspects 78 to 86, that    can specifically bind to HER2 with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   88. Amino acid sequence according to any of aspects 78 to 87, that    can specifically bind to HER2 with a rate of association    (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,    preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably    between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and    10⁷ M⁻¹ s⁻¹.-   89. Amino acid sequence according to any of aspects 78 to 88, that    can specifically bind to HER2 with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ S⁻¹.-   90. Amino acid sequence according to any of aspects 78 to 89, that    can specifically bind to HER2 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 μM.-   91. Amino acid sequence according to any of aspects 78 to 90, that    is a naturally occurring amino acid sequence (from any suitable    species) or a synthetic or semi-synthetic amino acid sequence.-   92. Amino acid sequence according to any of aspects 78 to 91, that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.-   93. Amino acid sequence according to any of aspects 78 to 92, that    is an immunoglobulin sequence.-   94. Amino acid sequence according to any of aspects 78 to 93, that    is a naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.-   95. Amino acid sequence according to any of aspects 78 to 94, that    is a humanized immunoglobulin sequence, a camelized immunoglobulin    sequence or an immunoglobulin sequence that has been obtained by    techniques such as affinity maturation.-   96. Amino acid sequence according to any of aspects 78 to 95, that    essentially consists of a light chain variable domain sequence (e.g.    a V_(L)-sequence); or of a heavy chain variable domain sequence    (e.g. a V_(H)-sequence).-   97, Amino acid sequence according to any of aspects 78 to 96, that    essentially consists of a heavy chain variable domain sequence that    is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   98. Amino acid sequence according to any of aspects 78 to 97, that    essentially consists of a domain antibody (or an amino acid sequence    that is suitable for use as a domain antibody), of a single domain    antibody (or an amino acid sequence that is suitable for use as a    single domain antibody), of a “dAb” (or an amino acid sequence that    is suitable for use as a dAb) or of a Nanobody® (including but not    limited to a V_(HH) sequence).-   99. Amino acid sequence according to any of aspects 78 to 98, that    essentially consists of a Nanobody®.-   100. Amino acid sequence according to any of aspects 78 to 99, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   101. Amino acid sequence according to any of aspects 78 to 100, that    essentially consists of a Nanobody® that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 2051-2325, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   102. Amino acid sequence according to any of aspects 78 to 101, that    essentially consists of a humanized Nanobody®.-   103. Amino acid sequence according to any of aspects 78 to 102, that    in addition to the at least one binding site for binding formed by    the CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.-   104. Nanobody that is directed against and/or that can specifically    bind to HER2.-   105. Nanobody according to aspect 104, wherein said Nanobody    competes with Herceptin® for binding to HER2.-   106. Nanobody according to any of aspects 104 or 105, wherein said    Nanobody inhibits and/or blocks binding of Herceptin® to HER2.-   107. Nanobody according to any of aspects 104 to 106, wherein said    Nanobody is directed against the Herceptin® binding site on HER2.-   108. Nanobody according to any of aspects 104 to 107, wherein said    Nanobody specifically binds to domain IV of HER2.-   109. Nanobody according to aspect 104, wherein said Nanobody    competes with Omnitarg for binding to HER2.-   110. Nanobody according to any of aspects 104 or 109, wherein said    Nanobody inhibits and/or blocks binding of Omnitarg to HER2.-   111. Nanobody according to any of aspects 109 or 110, wherein said    Nanobody is directed against the Omnitarg binding site on HER2.-   112. Nanobody according to any of aspects 109 to 111, wherein said    Nanobody specifically binds to domain II of HER2.-   113. Nanobody according to any of aspects 104 to 112, wherein said    Nanobody competes with Herceptin® and Omnitarg for binding to HER2.-   114. Nanobody according to any of aspects 104 to 113, wherein said    Nanobody inhibits and/or blocks binding of Herceptin® and Omnitarg    to HER2.-   115. Nanobody according to any of aspects 104 to 114, wherein said    Nanobody inhibits and/or blocks tumor cell proliferation.-   116. Nanobody according to any of aspects 104 to 115, wherein said    Nanobody inhibits, downregulates and/or blocks cell signalling.-   117. Nanobody according to any of aspects 104 to 116, wherein said    Nanobody induces apoptosis in tumor cells.-   118. Nanobody according to any of aspects 104 to 117, wherein said    Nanobody inhibits and/or blocks heterodimerization between ERBB    receptors.-   119. Nanobody according to any of aspects 104 to 118, wherein said    Nanobody inhibits and/or blocks ligand activation of an ERbB    hetero-oligomer comprising HER2 and HER3, HER4 or EGFR.-   120. Nanobody according to any of aspects 104 to 119, wherein said    Nanobody inhibits and/or blocks tumor vascularisation.-   121. Nanobody according to any of aspects 104 to 120, wherein said    Nanobody recruits immune effector cells such as macrophages and    monocytes to the tumor.-   122. Nanobody according to any of aspects 104 to 121, wherein said    Nanobody inhibits and/or blocks TNF induced signalling and/or cell    proliferation.-   123. Nanobody according to any of aspects 104 to 122, wherein said    Nanobody downregulates HER2 levels and/or downregulates    HER2-mediated signalling pathways.-   124. Nanobody according to any of aspects 104 to 123, wherein said    Nanobody inhibits and/or blocks metalloproteinase-mediated HER2    ectodomain shedding.-   125. Nanobody according to any of aspects 104 to 124, wherein said    Nanobody downregulates and/or blocks ligand-mediated ErbB    signalling.-   126. Nanobody according to any of aspects 104 to 125, wherein said    Nanobody inhibits and/or blocks HER2 ectodomain cleavage.-   127. Nanobody according to any of aspects 104 to 126, wherein said    Nanobody inhibits and/or blocks Heregulin-mediated activation of    MAPK/Erk1/2.-   128. Nanobody according to any of aspects 104 to 127, wherein said    Nanobody inhibits and/or blocks PI3K/Akt signalling.-   129. Nanobody according to any of aspects 104 to 128, wherein said    Nanobody modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Herceptin®.-   130. Nanobody according to any of aspects 104 to 129, wherein said    Nanobody modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Omnitarg.-   131. Nanobody according to any of aspects 104 to 130, that is in    essentially isolated form.-   132. Nanobody according to any of aspects 104 to 131, that can    specifically bind to HER2 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   133. Nanobody according to any of aspects 104 to 132, that can    specifically bind to HER2 with a rate of association (k_(on)-rate)    of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³    M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷    M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹.-   134. Nanobody according to any of aspects 104 to 133, that can    specifically bind to HER2 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹.-   135. Nanobody according to any of aspects 104 to 134, that can    specifically bind to HER2 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 μM.-   136. Nanobody according to any of aspects 104 to 135, that is a    naturally occurring Nanobody (from any suitable species) or a    synthetic or semi-synthetic Nanobody.-   137. Nanobody according to any of aspects 104 to 136, that is a    V_(HH) sequence, a partially humanized V_(HH) sequence, a fully    humanized V_(HH) sequence, a camelized heavy chain variable domain    or a Nanobody that has been obtained by techniques such as affinity    maturation.-   138. Nanobody according to any of aspects 104 to 137, that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   139. Nanobody according to any of aspects 104 to 138, that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 2051-2325, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-3.-   140. Nanobody according to any of aspects 104 to 139, in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775.-   141. Nanobody according to any of aspects 104 to 140, in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 401-675;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 401-675;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 401-675;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 951-1225;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 951-1225;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 951-1225;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 1501-1775;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 1501-1775;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 1501-1775.-   142. Nanobody according to any of aspects 104 to 141, in which the    CDR sequences have at least 70% amino acid identity, preferably at    least 80% amino acid identity, more preferably at least 90% amino    acid identity, such as 95% amino acid identity or more or even    essentially 100% amino acid identity with the CDR sequences of at    least one of the amino acid sequences of SEQ ID NO's: 2051-2325.-   143. Nanobody according to any of aspects 104 to 1.42, which is a    partially humanized Nanobody.-   144. Nanobody according to any of aspects 104 to 143, which is a    fully humanized Nanobody.-   145. Nanobody according to any of aspects 104 to 144, that is chosen    from the group consisting of SEQ ID NO's: 2051-2325 or from the    group consisting of amino acid sequences that have more than 80%,    preferably more than 90%, more preferably more than 95%, such as 99%    or more sequence identity (as defined herein) with at least one of    the amino acid sequences of SEQ ID NO's: 2051-2325.-   146. Nanobody according to any of aspects 104 to 145, that is chosen    from the group consisting of SEQ ID NO's: 2051-2325.-   147. Nanobody directed against HER2 that cross-blocks the binding of    at least one of the Nanobodies according to any of aspects 140 to    146 to HER2.-   148. Nanobody directed against HER2 that is cross-blocked from    binding to HER2 by at least one of the Nanobodies according to any    of aspects 140 to 146.-   149. Nanobody according to any of aspects 147 or 148, wherein the    ability of said Nanobody to cross-block or to be cross-blocked is    detected in a Biacore assay.-   150. Nanobody according to any of aspects 147 to 149, wherein the    ability of said Nanobody to cross-block or to be cross-blocked is    detected in an ELISA assay.-   151. Compound or construct, that comprises or essentially consists    of one or more amino acid sequences according to any of aspects 1 to    103 and/or one or more Nanobodies according to any of aspects 104 to    150, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.-   152. Compound or construct according to aspect 151, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.-   153. Compound or construct according to aspect 151, in which said    one or more linkers, if present, are one or more amino acid    sequences.-   154. Compound or construct according to any of aspects 151 to 153,    in which said one or more other groups, residues, moieties or    binding units are immunoglobulin sequences.-   155. Compound or construct according to any of aspects 151 to 154,    in which said one or more other groups, residues, moieties or    binding units are chosen from the group consisting of domain    antibodies, amino acid sequences that are suitable for use as a    domain antibody, single domain antibodies, amino acid sequences that    are suitable for use as a single domain antibody, “dAb”'s, amino    acid sequences that are suitable for use as a dAb, or Nanobodies.-   156. Compound or construct according to any of aspect 151 to 155, in    which said one or more amino acid sequences of the invention are    immunoglobulin sequences.-   157. Compound or construct according to any of aspects 151 to 156,    in which said one or more amino acid sequences of the invention are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or Nanobodies.-   158. Compound or construct, that comprises or essentially consists    of one or more Nanobodies according to any of aspects 104 to 150 and    in which said one or more other groups, residues, moieties or    binding units are Nanobodies.-   159. Compound or construct according to any of aspects 151 to 158,    which is a multivalent construct.-   160. Compound or construct according to aspect 159, that is chosen    from the group consisting of SEQ ID NO's: 2326-2330 or from the    group consisting of from amino acid sequences that have more than    80%, preferably more than 90%, more preferably more than 95%, such    as 99% or more sequence identity (as defined herein) with at least    one of the amino acid sequences of SEQ ID NO's: 2326-2330.-   161. Compound or construct according to any of aspects 151 to 160,    which is a multiparatopic construct.-   162. Compound or construct according to aspect 161, which is a    biparatopic or triparatopic construct.-   163. Compound or construct according to aspect 162, which comprises    at least one amino acid sequence directed against a first antigenic    determinant, epitope, part or domain of HER-2 and at least one amino    acid sequence directed against a second antigenic determinant,    epitope, part or domain of HER-2 different from the first antigenic    determinant, epitope, part or domain-   164. Biparatopic compound or construct according to aspect 163,    which is capable of simultaneously binding to said first antigenic    determinant, epitope, part or domain of HER2 and to said second    antigenic determinant, epitope, part or domain of HER2.-   165. Compound or construct according to any of aspects 151 to 164,    which combines two or more different modes of action each mediated    by one of its binding units, wherein each binding unit binds at a    different binding site of HER2.-   166. Compound or construct according to any of aspects 151 to 165,    which modulates, inhibits, downregulate and/or blocks two different    cell signaling pathways, each mediated by one of its binding units,    wherein each binding unit binds at a different binding site of HER2.-   167. Compound or construct according to aspect 151 to 166, wherein    said compound or construct competes with Herceptin® for binding to    HER2.-   168. Compound or construct according to any of aspects 151 to 167,    wherein said compound or construct inhibits and/or blocks binding of    Herceptin® to HER2.-   169. Compound or construct according to any of aspects 151 to 168,    wherein said compound or construct is directed against the    Herceptin® binding site on HER2.-   170. Compound or construct according to any of aspects 151 to 169,    wherein said compound or construct specifically binds to domain IV    of HER2.-   171. Compound or construct according to any of aspects 151 to 170,    wherein said compound or construct recruits immune effector cells    such as macrophages and monocytes to the tumor.-   172. Compound or construct according to any of aspects 151 to 171,    wherein said compound or construct downregulates HER2 levels and/or    downregulates HER2-mediated signalling pathways.-   173. Compound or construct according to any of aspects 151 to 172,    wherein said compound or construct inhibits and/or blocks    metalloproteinase-mediated. HER2 ectodomain shedding.-   174. Compound or construct according to any of aspects 151 to 173,    wherein said compound or construct modulates HER2 or HER2 mediated    signalling via the same mechanism of action as Herceptin®.-   175. Compound or construct according to aspect 151 to 174, wherein    said compound or construct competes with Omnitarg for binding to    HER2.-   176. Compound or construct according to any of aspects 151 to 175,    wherein said compound or construct inhibits and/or blocks binding of    Omnitarg to HER2.-   177. Compound or construct according to any of aspects 151 to 176,    wherein said compound or construct is directed against the Omnitarg    binding site on HER2.-   178. Compound or construct according to any of aspects 151 to 177,    wherein said compound or construct specifically binds to domain II    of HER2.-   179. Compound or construct according to any of aspects 151 to 178,    wherein said compound or construct inhibits and/or blocks ligand    activation of an ERbB hetero-oligomer comprising HER2 and HER3, HER4    or EGFR.-   180. Compound or construct according to any of aspects 151 to 179,    wherein said compound or construct is directed against the    Herceptin® binding site on HER2 and against the Omnitarg binding    site on HER2.-   181. Compound or construct according to any of aspects 151 to 180,    wherein said compound or construct modulates HER2 or HER2 mediated    signalling via the same mechanism of action as Omnitarg.-   182. Compound or construct according to any of aspects 151 to 181,    wherein said compound or construct competes with Herceptin® and    Omnitarg for binding to HER2.-   183. Compound or construct according to any of aspects 151 to 182,    wherein said compound or construct inhibits and/or blocks binding of    Herceptin® and Omnitarg to HER2.-   184. Compound or construct according to any of aspects 151 to 183,    which specifically binds the Omnitarg binding site on HER2 and to    the Herceptin® binding site on HER2.-   185. Compound or construct according to any of aspects 151 to 184,    which specifically binds to domain IV and domain II of HER2.-   186. Biparatopic compound or construct according to any of aspects    151 to 185, which can simultaneously bind the Omnitarg binding site    on HER2 and to the Herceptin® binding site on HER2.-   187. Compound or construct according to any of aspects 151 to 186,    which down-regulates HER2 levels while at the same time inhibiting    and/or blocking heterodimerization between ERBB receptors.-   188. Compound or construct according to any of aspects 151 to 187,    which combines the mode of action of Herceptin® and Omnitarg.-   189. Compound or construct according to any of aspects 151 to 188,    wherein said compound or construct inhibits and/or blocks tumor cell    proliferation.-   190. Compound or construct according to any of aspects 151 to 189,    wherein said compound or construct inhibits, downregulates and/or    blocks cell signalling.-   191. Compound or construct according to any of aspects 151 to 190,    wherein said compound or construct induces apoptosis in tumor cells.-   192. Compound or construct according to any of aspects 151 to 191,    wherein said compound or construct inhibits and/or blocks    heterodimerization between ERBB receptors.-   193. Compound or construct according to any of aspects 151 to 192,    wherein said compound or construct inhibits and/or blocks tumor    vascularisation.-   194. Compound or construct according to any of aspects 151 to 193,    wherein said compound or construct inhibits and/or blocks TNF    induced signalling and/or cell proliferation.-   195. Compound or construct according to any of aspects 151 to 194,    wherein said compound or construct inhibits, downregulates and/or    blocks ligand-mediated ErbB signalling.-   196. Compound or construct according to any of aspects 151 to 195,    wherein said compound or construct inhibits and/or blocks HER2    ectodomain cleavage.-   197. Compound or construct according to any of aspects 151 to 196,    wherein said compound or construct inhibits and/or blocks    Heregulin-mediated activation of MAPK/Erk1/2.-   198. Compound or construct according to any of aspects 151 to 197,    wherein said compound or construct inhibits and/or blocks PI3K/Akt-   199. Compound or construct according to any of aspects 151 to 198,    wherein said compound or construct is linked to an Fc portion.-   200. Compound or construct that comprises an Fc portion that is    linked, optionally via a suitable linker or hinge region, to a pair    of first amino acid sequences according to any of aspects 1 to 103    or Nanobodies according to any of aspects 104 to 150, which are    linked, optionally via a suitable linker, to a pair of second amino    acid sequences according to any of aspects 1 to 103 or Nanobodies    according to any of aspects 104 to 150, wherein:    -   both of the first amino acid sequences according to any of        aspects 1 to 103 or Nanobodies according to any of aspects 104        to 150 are directed against a first epitope, antigenic        determinant, part, domain or subunit on HER2; and    -   both of the second amino acid sequences according to any of        aspects 1 to 103 or Nanobodies according to any of aspects 104        to 150 are directed against a second epitope, antigenic        determinant, part, domain or subunit on HER2 which is different        from said first epitope, antigenic determinant, part, domain or        subunit.-   201. Compound or construct according to any of aspects 199 or 200,    wherein the first amino acid sequences according to any of aspects 1    to 103 or Nanobodies according to any of aspects 104 to 150 are    directed against the Herceptin® binding site on HER-2 and/or are    amino acid sequences or Nanobodies that are capable of competing    with Herceptin® for binding to HER-2.-   202. Compound or construct according to any of aspects 199 to 201,    wherein the first amino acid sequences according to any of aspects 1    to 103 or Nanobodies according to any of aspects 104 to 150 are    directed against the Omnitarg® binding site on HER-2 and/or are    amino acid sequences or Nanobodies that are capable of competing    with Omnitarg® for binding to HER-2.-   203. Compound or construct according to any of aspects 199 to 202,    wherein the first amino acid sequences according to any of aspects 1    to 103 or Nanobodies according to any of aspects 104 to 150 are    directed against the Herceptin® binding site on HER-2 and/or are    amino acid sequences or Nanobodies that are capable of competing    with Herceptin® for binding to HER-2 and the second amino acid    sequences according to any of aspects 1 to 103 or Nanobodies    according to any of aspects 104 to 150 are directed against the    Omnitarg® binding site on HER-2 and/or are amino acid sequences or    Nanobodies that are capable of competing with Omnitarg® for binding    to HER-2 (or visa versa).-   204. Compound or construct according to any of aspects 151 to 203,    that comprises or that is chosen from the group consisting of SEQ ID    NO's: 2336-2390 or from the group consisting of from amino acid    sequences that have more than 80%, preferably more than 90%, more    preferably more than 95%, such as 99% or more sequence identity (as    defined herein) with at least one of the amino acid sequences of SEQ    ID NO's: 2336-2390.-   205. Compound or construct according to any of aspects 151 to 204,    which is a multispecific construct.-   206. Compound or construct according to any of aspects 151 to 205,    which has an increased half-life, compared to the corresponding    amino acid sequence according to any of aspects 1 to 103 per se or    Nanobody according to any of aspects 104 to 150 per se,    respectively.-   207. Compound or construct according to any of aspects 151 to 206,    in which said one or more other groups, residues, moieties or    binding units provide the compound or construct with increased    half-life, compared to the corresponding amino acid sequence    according to any of aspects 1 to 103 per se or Nanobody according to    any of aspects 104 to 150 per se, respectively.-   208. Compound or construct according to aspect 207, in which said    one or more other groups, residues, moieties or binding units that    provide the compound or construct with increased half-life is chosen    from the group consisting of serum proteins or fragments thereof,    binding units that can bind to serum proteins, an Fc portion, and    small proteins or peptides that can bind to serum proteins.-   209. Compound or construct according to aspect 207, in which said    one or more other groups, residues, moieties or binding units that    provide the compound or construct with increased half-life is chosen    from the group consisting of human serum albumin or fragments    thereof.-   210. Compound or construct according to aspect 207, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life are    chosen from the group consisting of binding units that can bind to    serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).-   211. Compound or construct according to aspect 207, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or Nanobodies that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).-   212. Compound or construct according to aspect 207, in which said    one or more other groups, residues, moieties or binding units that    provides the compound or construct with increased half-life is a    Nanobody that can bind to serum albumin (such as human serum    albumin) or a serum immunoglobulin (such as IgG).-   213. Compound or construct according to any of aspects 206 to 212,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding amino acid sequence according to any of aspects 1 to    103 per se or Nanobody according to any of aspects 104 to 150 per    se, respectively.-   214. Compound or construct according to any of aspects 206 to 213,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding amino acid sequence according to any    of aspects 1 to 103 per se or Nanobody according to any of aspects    104 to 150 per se, respectively.-   215. Compound or construct according to any of aspects 206 to 214,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, of at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).-   216. Compound or construct according to any of aspects 151 to 215,    in which said one or more other groups, residues, moieties or    binding units are one or more constant domains.-   217. Compound or construct according to aspect 216, wherein said one    or more constant domains are two constant domains that can be used    as part of/to form an Fc portion.-   218. Compound or construct according to any of aspects 216 or 217,    wherein said one or more constant domains are three constant domains    that can be used as part of/to form an Fc portion.-   219. Compound or construct according to any of aspects 216 to 218,    wherein said one or more constant domains confer one or more    effector functions to the compound or construct.-   220. Compound or construct according to any of aspects 216 to 219,    wherein said one or more constant domains confer the ability to bind    to one or more Fc receptors.-   221. Compound or construct according to any of aspects 216 to 220,    in which said one or more amino acid sequences or Nanobodies are    linked to the Fc portion via a linker or hinge region.-   222. Compound or construct according to any of aspects 216 to 221,    wherein said one or more constant domains are from IgG (e.g. from    IgG1, IgG2, IgG3 or IgG4).-   223. Compound or construct according to any of aspects 216 to 221,    wherein said one or more constant domains are from human Ig such as    IgA, IgD or IgM.-   224. Compound or construct according to any of aspects 216 to 221,    wherein said one or more constant domains are from IgE.-   225. Compound or construct that comprises two amino acids according    to any of aspects 1 to 103 or Nanobodies according to any of aspects    104 to 150 and an Fc portion that is capable of effecting one or    more IgE-mediated immune responses and/or that is capable of binding    to either the FcεRI receptor and/or the FcεRII receptor.-   226. Compound or construct according to aspect 225, comprising two    polypeptide chains, in which each polypeptide chain comprises at    least one amino acid sequence according to any of aspects 1 to 103    or at least one Nanobody according to any of aspects 104 to 150 that    is linked, optionally via a suitable linker or hinge region, to one    or more constant domains, in which the constant domains from the    first polypeptide chain and the second polypeptide chains together    form an Fc portion that is capable of effecting one or more    IgE-mediated immune responses and/or that is capable of binding to    either the FcεRI receptor and/or the FcεRII receptor.-   227. Compound or construct according to any of aspects 224 to 226,    in which the Fc portion is capable of binding to FcεRI with an    affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹,    preferably better than 10⁸ M⁻¹, more preferably better than 10⁹ M⁻¹,    such as with a K_(a) value of about 10¹⁰ M⁻¹ or 10¹¹ M⁻¹.-   228. Compound or construct according to any of aspects 224 to 227,    in which the Fc portion is capable of binding to FcεRII with an    affinity (expressed as the K_(a) value) better than 10⁶ M⁻¹,    preferably better than 10⁷ M⁻¹, such as with a K_(a) value of about    10⁸ M⁻¹.-   229. Compound or construct according to any of aspects 224 to 228,    in which the Fc portion comprises at least one or more parts,    fragments, amino acid stretches or domains of the Fc portion of IgE.-   230. Compound or construct according to any of aspects 224 to 229,    in which the Fc portion comprises at least one or more of those    parts, fragments, amino acid stretches or domains of the Fc portion    of IgE that allow IgE to bind to its receptors.-   231. Compound or construct according to any of aspects 224 to 230,    in which the Fc portion at least comprises C_(ε)4 (or a suitable    part of fragment thereof), and optionally also comprises C_(ε)3 (or    a suitable part of fragment thereof) and/or C_(ε)2 (or a suitable    part of fragment thereof).-   232. Compound or construct according to any of aspects 224 to 231,    in which the Fc portion essentially consist of three constant    domains.-   233. Compound or construct according to any of aspects 224 to 232,    in which the parts, fragments, amino acid stretches or domains that    make up the IgE-derived Fc portion are preferably all or essentially    all derived from human Fc portions.-   234. Compound or construct according to any of aspects 224 to 233,    in which the IgE-derived Fc portion is from human IgE.-   235. Monovalent construct, comprising or essentially consisting of    one amino acid sequence according to any of aspects 1 to 103 and/or    one Nanobody according to any of aspects 104 to 150.-   236. Monovalent construct according to aspect 235, in which said    amino acid sequence is chosen from the group consisting of domain    antibodies, amino acid sequences that are suitable for use as a    domain antibody, single domain antibodies, amino acid sequences that    are suitable for use as a single domain antibody, “dAb”'s, amino    acid sequences that are suitable for use as a dAb, or Nanobodies.-   237. Monovalent construct, comprising or essentially consisting of    one Nanobody according to any of aspects 104 to 150.-   238. Monovalent construct, that is chosen from the group consisting    of SEQ ID NO's: 2051-2325 or from the group consisting of amino acid    sequences that have more than 80%, preferably more than 90%, more    preferably more than 95%, such as 99% or more sequence identity (as    defined herein) with at least one of the amino acid sequences of SEQ    ID NO's: 2051-2325.-   239. Use of a monovalent construct according to any of aspects 235    to 238, in preparing a multivalent compound or construct according    to any of aspects 151 to 234.-   240. Use of a monovalent construct according to aspect 239, in    preparing a multiparatopic construct such as a biparatopic    construct.-   241. Use of a monovalent construct according to any of aspects 239    or 240, wherein the monovalent construct is used as a binding domain    or binding unit in preparing a multivalent construct comprising two    or more binding units.-   242. Use of a monovalent construct according to any of aspects 239    to 241, in preparing a multivalent construct that exhibits    intramolecular binding compared to intermolecular binding.-   243. Use of a monovalent construct according to any of aspects 239    to 242, as a binding domain or binding unit in preparing a    multivalent construct, wherein the binding domains or binding units    are linked via a linker such that the multivalent construct    preferably exhibits intramolecular binding compared to    intermolecular binding.-   244. Use of a monovalent construct according to any of aspects 239    to 243, wherein the monovalent construct is directed against the    Omnitarg binding site on HER2 and/or is capable of competing with    Omnitarg for binding to HER-2.-   245. Use of a monovalent construct according to any of aspects 239    to 244, wherein the monovalent construct is directed against domain    II of HER2.-   246. Use of a monovalent construct according to any of aspects 239    to 245, wherein the monovalent construct is directed against the    middle of domain II of HER2.-   247. Use of a monovalent construct according to any of aspects 239    to 243, wherein the monovalent construct is directed against the    Herceptin® binding site on HER2.-   248. Use of a monovalent construct according to any of aspects 239    to 243 or 247, wherein the monovalent construct is directed against    domain IV of HER2.-   249. Use of a monovalent construct according to any of aspects 239    to 243 or 247 to 248, wherein the monovalent construct is directed    against the C-terminus of domain IV of HER2.-   250. Use of two monovalent constructs according to any of aspects    239 to 249, wherein a first monovalent construct is directed against    the Omnitarg binding site on HER2 (and in particular against domain    II of HER2, and more in particular against the middle of domain II    of HER2) and/or is capable of competing with Omnitarg for binding to    HER-2 and wherein the second monovalent construct is directed    against the Herceptin® binding site on HER2 (and in particular    against domain IV of HER2, and more in particular against the    C-terminus of domain IV of HER2) and/or is capable of competing with    Herceptin® for binding to HER-2.-   251. Nucleic acid or nucleotide sequence, that encodes an amino acid    sequence according to any of aspects 1 to 103, a Nanobody according    to any of aspects 104 to 150, a compound or construct according to    any of aspects 151 to 234 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same, or a monovalent construct according to any of aspects 235 to    238.-   252. Nucleic acid or nucleotide sequence according to aspect 251,    that is in the form of a genetic construct.-   253. Use of a nucleic acid or nucleotide sequence according to    aspect 251, that encodes a monovalent construct according to any of    aspects 235 to 238, for the preparation of a genetic construct that    encodes a multivalent construct according to any of aspects 151 to    234.-   254. Use of a nucleic acid or nucleotide sequence according to    aspect 253, wherein the genetic construct encodes a multiparatopic    (such as a biparatopic) construct.-   255. Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an amino acid sequence    according to any of aspects 1 to 103, a Nanobody according to any of    aspects 104 to 150, a compound or construct according to any of    aspects 151 to 234 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same, or a monovalent construct according to any of aspects 235 to    238; and/or that comprises a nucleic acid or nucleotide sequence    according to aspect 251, or a genetic construct according to aspect    252.-   256. Method for producing an amino acid sequence according to any of    aspects 1 to 103, a Nanobody according to any of aspects 104 to 150,    a compound or construct according to any of aspects 151 to 234 that    is such that it can be obtained by expression of a nucleic acid or    nucleotide sequence encoding the same, or a monovalent construct    according to any of aspects 235 to 238, said method at least    comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect 251, or a genetic construct        according to aspect 252    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects 1 to 103, the Nanobody according to any of        aspects 104 to 150, the compound or construct according to any        of aspects 151 to 234 that is such that it can be obtained by        expression of a nucleic acid or nucleotide sequence encoding the        same, or the monovalent construct according to any of aspects        235 to 238 thus obtained.-   257. Method for producing an amino acid sequence according to any of    aspects 1 to 103, a Nanobody according to any of aspects 104 to 150,    a compound or construct according to any of aspects 151 to 234 that    is such that it can be obtained by expression of a nucleic acid or    nucleotide sequence encoding the same, or a monovalent construct    according to any of aspects 235 to 238, said method at least    comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect 255 under conditions that are such that said host or        host cell expresses and/or produces at least one amino acid        sequence according to any of aspects 1 to 103, Nanobody        according to any of aspects 104 to 150, compound or construct        according to any of aspects 151 to 234 that is such that it can        be obtained by expression of a nucleic acid or nucleotide        sequence encoding the same, or a monovalent construct according        to any of aspects 235 to 238,    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects 1 to 103, the Nanobody according to any of        aspects 104 to 150, the compound or construct according to any        of aspects 151 to 234 that is such that it can be obtained by        expression of a nucleic acid or nucleotide sequence encoding the        same, or the monovalent construct according to any of aspects        235 to 238 thus obtained.-   258. Method for preparing and/or generating a multiparatopic (such    as e.g. biparatopic, triparatopic, etc.) construct according to any    of aspects 161 to 204, said method comprising at least the steps of:    -   a) providing a nucleic acid sequence according to aspect 251,        encoding a first HER2 binding amino acid sequence, fused to a        set, collection or library of nucleic acid sequences encoding        amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode a second amino        acid sequence that can bind to and/or has affinity for an        antigenic determinant on HER2 different from the antigenic        determinant recognized by the first HER2 binding amino acid        sequence;    -   and    -   c) isolating the nucleic acid sequence encoding an HER2 binding        amino acid sequence fused to the nucleic acid sequence obtained        in b), followed by expressing the encoded construct.-   259. Method for preparing and/or generating a biparatopic construct    according to any of aspects 161 to 204, said method comprising at    least the steps of:    -   a) providing a set, collection or library of nucleic acid        sequences, in which each nucleic acid sequence in said set,        collection or library encodes a fusion protein that comprises a        first amino acid sequence that can bind to and/or has affinity        for a first antigenic determinant, part, domain or epitope on        HER2 that is fused (optionally via a linker sequence) to a        second amino acid sequence, in which essentially each second        amino acid sequence (or most of these) is a different member of        a set, collection or library of different amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for a second        antigenic determinant, part, domain or epitope on HER2 different        from the first antigenic determinant, part, domain or epitope on        HER-2;    -   and    -   c) isolating the nucleic acid sequences that encode an amino        acid sequence that can bind to and/or has affinity for a second        antigenic determinant, part, domain or epitope on HER2 different        from the first antigenic determinant, part, domain or epitope on        HER-2, obtained in b), optionally followed by expressing the        encoded amino acid sequence.-   260. Method according to aspect 259, wherein the first amino acid is    also encoded by a set, collection or library of nucleic acid    sequences and wherein, in step b), said set, collection or library    of nucleic acid sequences is screened for nucleic acid sequences    that encode an amino acid sequence that can bind to and/or has    affinity for the first antigenic determinant, part, domain or    epitope on HER2.-   261. Method according to aspect 260, wherein the screening in    step b) is performed in a single step.-   262. Method according to aspect 260, wherein the screening in    step b) is performed in subsequent steps.-   263. Method according to any of aspects 258 to 262, wherein the    first amino acid sequence used in step a) is preferably such    that (i) it can bind to and/or has affinity for the Herceptin®    binding site on HER2 (and in particular domain IV of HER2, more in    particular the C-terminus of domain IV of HER2) and/or (ii) competes    with Herceptin® for binding to HER-2.-   264. Method according to any of aspects 258 to 263, wherein in step    b), the set, collection or library of nucleic acid sequences is    screened for nucleic acid sequences that encode (i) an amino acid    sequence that can bind to and/or has affinity for the Omnitarg    binding site on HER2 (and in particular domain TI of HER2, more in    particular the middle of domain II of HER2) and/or (ii) an amino    acid sequence that can compete with Omnitarg or Omnitarg Fab for    binding to HER-2.-   265. Method according to any of aspects 258 to 264, wherein the    first amino acid sequence used in step a) is preferably such    that (i) it can bind to and/or has affinity for the Omnitarg binding    site on HER2 (and in particular domain II of HER2, more in    particular the middle of domain H of HER2) and/or (ii) competes with    Omnitarg for binding to HER-2.-   266. Method according to any of aspects 258 to 265, wherein in step    b), the set, collection or library of nucleic acid sequences is    screened for nucleic acid sequences that encode (i) an amino acid    sequence that can bind to and/or has affinity for the Herceptin®    binding site on HER2 (and in particular domain IV of HER2, more in    particular the C-terminus of domain IV of HER2) and/or (ii) an amino    acid sequence that can compete with Herceptin® for binding to HER-2.-   267. Method according to any of aspects 258 to 266, wherein in step    b), the set, collection or library of nucleic acid sequences is    screened for nucleic acid sequences that both (i) encode an amino    acid sequence that can bind to and/or has affinity for the Omnitarg    binding site on HER2 (and in particular domain II of HER2, more in    particular the middle of domain II of HER2) and/or that can compete    with Omnitarg or Omnitarg Fab for binding to HER-2 and that    also (ii) encode an amino acid sequence that can bind to and/or has    affinity for the Herceptin® binding site on HER2 (and in particular    domain IV of HER2, more in particular the C-terminus of domain IV of    HER2) and/or that can compete with Herceptin® for binding to HER-2.-   268. Method according to aspect 267, wherein the screening in    step b) is performed in a single step.-   269. Method according to aspect 267, wherein the screening in    step b) is performed in a subsequent steps.-   270. Method according to any of aspects 258 to 269, wherein the    screening in step b) is performed in the presence of Herceptin®    and/or Omnitarg.-   271. Method for screen for suitable and/or optimal linker lengths    for linking a first and a second amino acid sequence in a    biparatopic constructs according to any of aspects 161 to 204,    wherein said method comprises at least the steps of:    -   d) providing a set, collection or library of nucleic acid        sequences, in which each nucleic acid sequence in said set,        collection or library encodes a fusion protein that comprises a        first amino acid sequence that can bind to and/or has affinity        for a first antigenic determinant, part, domain or epitope on        HER2 that is fused via a linker sequence to a second amino acid        sequence that has can bind to and/or has affinity for a second        antigenic determinant, part, domain or epitope on HER2 (which        may be the same or different as the first antigenic determinant,        part, domain or epitope on HER2), in which essentially each        nucleic acid sequence (or most of these) encodes a fusion        protein with a different linker sequence so as to provide a set,        collection or library encoding different fusion proteins;    -   e) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for the first and        second antigenic determinant, part, domain or epitope on HER2;    -   and    -   f) isolating the nucleic acid sequences that encode an amino        acid sequence that can bind to and/or has affinity for the first        and second antigenic determinant, part, domain or epitope on        HER2, optionally followed by expressing the encoded amino acid        sequence.-   272. Method according to aspect 271, wherein the first amino acid    sequence is an amino acid sequence that can bind to and/or has    affinity for the Omnitarg binding site on HER2 (and may in    particular domain II of HER2, more in particular the middle of    domain II of HER2) and/or that can compete with Omnitarg or Omnitarg    Fab.-   273. Method according to aspect 271, wherein the second amino acid    sequence is an amino acid sequence that can bind to and/or has    affinity for the Herceptin® binding site on HER2 (and in particular    domain IV of HER2, more in particular the C-terminus of domain IV of    HER2) and/or that can compete with Herceptin® for binding to HER-2.-   274. Method according to aspect 271, wherein the first amino acid    sequence is an amino acid sequence that can bind to and/or has    affinity for the Omnitarg binding site on HER2 (and may in    particular domain. II of HER2, more in particular the middle of    domain II of HER2) and/or that can compete with Omnitarg or Omnitarg    Fab and wherein the second amino acid sequence is an amino acid    sequence that can bind to and/or has affinity for the Herceptin®    binding site on HER2 (and in particular domain IV of HER2, more in    particular the C-terminus of domain IV of HER2) and/or that can    compete with Herceptin® for binding to HER-2.-   275. Method according to any of aspects 271 to 274, wherein the    screening in step b) is performed in a single step.-   276. Method according to any of aspects 271 to 274, wherein the    screening in step b) is performed in subsequent steps.-   277. Method according to any of aspects 271 to 276, wherein the    screening in step b) is performed in the presence of Herceptin®    and/or Omnitarg.-   278. Method for preparing and/or generating biparatopic constructs    according to any of aspects 161 to 204, said method comprising at    least the steps of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for a set, collection or library of nucleic acid        sequences that encode an amino acid sequence that can bind to        and/or has affinity for HER2;    -   c) ligating said set, collection or library of nucleic acid        sequences that encode an amino acid sequence that can bind to        and/or has affinity for HER2 to another nucleic acid sequence        that encodes an amino acid sequence that can bind to and/or has        affinity for HER2 (e.g. a nucleic acid sequence that encodes an        amino acid sequence that competes with Herceptin® for binding        HER2);    -   and    -   d) from the set, collection or library of nucleic acid sequences        obtained in c), isolating the nucleic acid sequences encoding a        biparatopic amino acid sequence that can bind to and/or has        affinity for HER2 (and e.g. further selecting for nucleic acid        sequences that encode a biparatopic amino acid sequence that        antagonizes with higher potency compared to the monovalent amino        acid sequences), followed by expressing the encoded amino acid        sequence.-   279. Method for preparing and/or generating biparatopic constructs    according to any of aspects, said method comprising at least the    steps of:    -   a) providing a first set, collection or library of nucleic acid        sequences encoding amino acid sequences;    -   b) screening said first set, collection or library of nucleic        acid sequences for a nucleic acid sequence that encodes an amino        acid sequence that can bind to and/or has affinity for a first        antigenic determinant, part, domain or epitope on HER2;    -   c) ligating the nucleic acid sequence encoding said amino acid        sequence that can bind to and/or has affinity for a first        antigenic determinant, part, domain or epitope on HER2 obtained        in b) to another set, collection or library of nucleic acid        sequences encoding amino acid sequences to obtain a set,        collection or library of nucleic acid sequences that encode        fusion proteins;    -   d) screening said set, collection or library of nucleic acid        sequences obtained in step c) for a nucleic acid sequence that        encodes an amino acid sequence that can bind to and has affinity        for a second antigenic determinant, part, domain or epitope on        HER2 which is the same or different from the first antigenic        determinant, part, domain or epitope on HER-2;    -   and    -   e) isolating the nucleic acid sequence that encodes an amino        acid sequence that can bind to and/or has affinity for the first        and second antigenic determinant, part, domain or epitope on        HER2, optionally followed by expressing the encoded amino acid        sequence.-   280. Method according to aspect 279, wherein in step b), the set,    collection or library of nucleic acid sequences is screened for    nucleic acid sequences that encode a first amino acid sequence    that (i) can bind to and/or has affinity for the Herceptin® binding    site on HER2 (and in particular domain IV of HER2, more in    particular the C-terminus of domain IV of HER2) and/or (ii) competes    with Herceptin® for binding to HER-2.-   281. Method according to aspect 279, wherein in step d), the set,    collection or library of nucleic acid sequences is screened for    nucleic acid sequences that encode a second amino acid sequence    that (i) can bind to and/or has affinity for the Omnitarg binding    site on HER2 (and in particular domain. II of HER2, more in    particular the middle of domain II of HER2) and/or (ii) that can    compete with Omnitarg or Omnitarg Fab for binding to HER-2.-   282. Method according to aspect 279, wherein in step b), the set,    collection or library of nucleic acid sequences is screened nucleic    acid sequences that encode a first amino acid sequence that (i) can    bind to and/or has affinity for the Herceptin® binding site on HER2    (and in particular domain IV of HER2, more in particular the    C-terminus of domain IV of HER2) and/or (ii) competes with    Herceptin® for binding to HER-2 and wherein in step d), the set,    collection or library of nucleic acid sequences is screened for    nucleic acid sequences that encode a second amino acid sequence    that (i) can bind to and/or has affinity for the Omnitarg binding    site on HER2 (and in particular domain II of HER2, more in    particular the middle of domain II of HER2) and/or (ii) that can    compete with Omnitarg or Omnitarg Fab for binding to HER-2 (or visa    versa).-   283. Method according to aspect 279, wherein in step b), the set,    collection or library of nucleic acid sequences is screened nucleic    acid sequences that encode a first amino acid sequence that (i) can    bind to and/or has affinity for the Omnitarg binding site on HER2    (and in particular domain II of HER2, more in particular the middle    of domain II of HER2) and/or (ii) competes with Omnitarg for binding    to HER-2.-   284. Method according to aspect 279, wherein in step d), the set,    collection or library of nucleic acid sequences is screened for    nucleic acid sequences that encode a second amino acid sequence    that (i) can bind to and/or has affinity for the Herceptin® binding    site on HER2 (and in particular domain IV of HER2, more in    particular the C-terminus of domain IV of HER2) and/or (ii) that can    compete with Herceptin® for binding to HER-2.-   285. Method according to aspect 279, wherein in step b), the set,    collection or library of nucleic acid sequences is screened nucleic    acid sequences that encode a first amino acid sequence that (i) can    bind to and/or has affinity for the Omnitarg binding site on HER2    (and in particular domain H of HER2, more in particular the middle    of domain II of HER2) and/or (ii) competes with Omnitarg for binding    to HER-2 and wherein in step d), the set, collection or library of    nucleic acid sequences is screened for nucleic acid sequences that    encode a second amino acid sequence that (i) can bind to and/or has    affinity for the Herceptin® binding site on HER2 (and in particular    domain IV of HER2, more in particular the C-terminus of domain. IV    of HER2) and/or (ii) that can compete with Herceptin® for binding to    HER-2.-   286. Method according to aspect 285, wherein the screening in    steps b) and/or d) is performed in the presence of Herceptin® and/or    Omnitarg.-   287. Method for preparing and/or generating a biparatopic constructs    according to any of aspects 161 to 204, said method comprising at    least the steps of linking two or more monovalent amino acid    sequences or monovalent construct according to any of aspects 235 to    238 and for example one or more linkers.-   288. Method according to aspect 287, comprising the steps of:    -   a) linking two or more nucleic acid sequences according to        aspect 251, encoding a monovalent construct according to any of        aspects 235 to 238 (and also for example nucleic acids encoding        one or more linkers and further one or more further elements of        genetic constructs known per se) to obtain a genetic construct        according to aspect 252;    -   b) expressing, in a suitable host cell or host organism or in        another suitable expression system, the genetic construct        obtained in a)    -   optionally followed by:    -   c) isolating and/or purifying the biparatopic constructs        according to any of aspects 161 to 204 thus obtained.-   289. Composition, comprising at least one amino acid sequence    according to any of aspects 1 to 103, Nanobody according to any of    aspects 104 to 150, compound or construct according to any of    aspects 151 to 234, monovalent construct according to any of aspects    235 to 238, or nucleic acid or nucleotide sequence according to    aspects 251 to 252.-   290. Composition according to aspect 289, which is a pharmaceutical    composition.-   291. Composition according to aspect 289, which is a pharmaceutical    composition, that further comprises at least one pharmaceutically    acceptable carrier, diluent or excipient and/or adjuvant, and that    optionally comprises one or more further pharmaceutically active    polypeptides and/or compounds.-   292. Method for the prevention and/or treatment of at least one    cancer and/or tumor, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 103,    Nanobody according to any of aspects 104 to 150, compound or    construct according to any of aspects 151 to 234, monovalent    construct according to any of aspects 235 to 238, or composition    according to aspect 290 or 291.-   293. Method for the prevention and/or treatment of at least one    disease or disorder that is associated with HER2, with its    biological or pharmacological activity, and/or with the biological    pathways or signalling in which HER2 is involved, said method    comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one amino acid sequence    according to any of aspects 1 to 103, Nanobody according to any of    aspects 104 to 150, compound or construct according to any of    aspects 151 to 234, monovalent construct according to any of aspects    235 to 238, or composition according to aspect 290 or 291.-   294. Method for the prevention and/or treatment of at least one    disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, an amino acid sequence    according to any of aspects 1 to 103, a Nanobody according to any of    aspects 104 to 150, a compound or construct according to any of    aspects 151 to 234, or a monovalent construct according to any of    aspects 235 to 238, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 103,    Nanobody according to any of aspects 104 to 150, compound or    construct according to any of aspects 151 to 234, monovalent    construct according to any of aspects 235 to 238, or composition    according to aspect 290 or 291.-   295. Method for immunotherapy, said method comprising administering,    to a subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects 1 to 103,    Nanobody according to any of aspects 104 to 150, compound or    construct according to any of aspects 151 to 234, monovalent    construct according to any of aspects 235 to 238, or composition    according to aspect 290 or 291.-   296. Use of an amino acid sequence according to any of aspects 1 to    103, a Nanobody according to any of aspects 104 to 150, a compound    or construct according to any of aspects 151 to 234, or a monovalent    construct according to any of aspects 235 to 238 in the preparation    of a pharmaceutical composition for prevention and/or treatment of    at least one cancer and/or tumor; and/or for use in one or more of    the methods according to aspects 292 to 295.-   297. An amino acid sequence according to any of aspects 1 to 103, a    Nanobody according to any of aspects 104 to 150, a compound or    construct according to any of aspects 151 to 234, or a monovalent    construct according to any of aspects 235 to 238 for use in the    prevention and/or treatment of at least one cancer and/or tumor;    and/or for use in one or more of the methods according to aspects    292 to 295.-   298. Part or fragment of an amino acid sequence according to any of    aspects 1 to 103, or of a Nanobody according to any of aspects 104    to 150.-   299. Part or fragment according to aspect 298, that can specifically    bind to HER2.-   300. Part or fragment according to aspect 299, wherein said part or    fragment competes with Herceptin® for binding to HER2.-   301. Part of fragment according to any of aspects 299 or 300,    wherein said part or fragment inhibits and/or blocks binding of    Herceptin® to HER2.-   302. Part or fragment according to any of aspects 299 to 301,    wherein said part or fragment is directed against the Herceptin®    binding site on HER2.-   303. Part of fragment according to any of aspects 299 to 302,    wherein said part or fragment specifically binds to domain IV of    HER2.-   304. Part of fragment according to aspect 299, wherein said part or    fragment competes with Omnitarg for binding to HER2.-   305. Part of fragment according to any of aspects 299 or 304,    wherein said part or fragment inhibits and/or blocks binding of    Omnitarg to HER2.-   306. Part of fragment according to any of aspects 304 or 305,    wherein said part or fragment is directed against the Omnitarg    binding site on HER2.-   307. Part of fragment according to any of aspects 304 to 306,    wherein said part or fragment specifically binds to domain II of    HER2.-   308. Part of fragment according to any of aspects 299 to 307,    wherein said part or fragment competes with Herceptin® and Omnitarg    for binding to HER2.-   309. Part of fragment according to any of aspects 299 to 308,    wherein said part or fragment inhibits and/or blocks binding of    Herceptin® and Omnitarg to HER2.-   310. Part of fragment according to any of aspects 299 to 309,    wherein said part or fragment inhibits and/or blocks tumor cell    proliferation.-   311. Part of fragment according to any of aspects 299 to 310,    wherein said part or fragment inhibits, downregulates and/or blocks    cell signalling.-   312. Part of fragment according to any of aspects 299 to 311,    wherein said part or fragment induces apoptosis in tumor cells.-   313. Part of fragment according to any of aspects 299 to 312,    wherein said part or fragment inhibits and/or blocks    heterodimerization between ERBB receptors.-   314. Part of fragment according to any of aspects 299 to 313,    wherein said part or fragment inhibits and/or blocks ligand    activation of an ERbB hetero-oligomer comprising HER2 and HER3, HER4    or EGFR.-   315. Part of fragment according to any of aspects 299 to 314,    wherein said part or fragment inhibits and/or blocks tumor    vascularisation.-   316. Part of fragment according to any of aspects 299 to 315,    wherein said part or fragment recruits immune effector cells such as    macrophages and monocytes to the tumor.-   317. Part of fragment according to any of aspects 299 to 316,    wherein said part or fragment inhibits and/or blocks TNF induced    signalling and/or cell proliferation.-   318. Part of fragment according to any of aspects 299 to 317,    wherein said part or fragment downregulates HER2 levels and/or    downregulates HER2-mediated signalling pathways.-   319. Part of fragment according to any of aspects 299 to 318,    wherein said part or fragment inhibits and/or blocks    metalloproteinase-mediated HER2 ectodomain shedding.-   320. Part of fragment according to any of aspects 299 to 319,    wherein said part or fragment inhibits, downregulates and/or blocks    ligand-mediated ErbB signalling.-   321. Part of fragment according to any of aspects 299 to 320,    wherein said part or fragment inhibits and/or blocks HER2 ectodomain    cleavage.-   322. Part of fragment according to any of aspects 299 to 321,    wherein said part or fragment inhibits and/or blocks    Heregulin-mediated activation of MAPK/Erk1/2.-   323. Part of fragment according to any of aspects 299 to 322,    wherein said part or fragment inhibits and/or blocks PI3K/Akt    signalling.-   324. Part of fragment according to any of aspects 299 to 323,    wherein said part or fragment modulates HER2 or HER2 mediated    signalling via the same mechanism of action as Herceptin®.-   325. Part of fragment according to any of aspects 299 to 324,    wherein said part or fragment modulates HER2 or HER2 mediated    signalling via the same mechanism of action as Omnitarg.-   326. Part of fragment according to any of aspects 299 to 325, that    can specifically bind to HER2 with a dissociation constant (K_(D))    of 10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   327. Part or fragment according to any of aspects 299 to 326, that    can specifically bind to HER2 with a rate of association    (k_(on)-rate) of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,    preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably    between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and    10⁷ M⁻¹ s⁻¹.-   328. Part or fragment according to any of aspects 299 to 327, that    can specifically bind to HER2 with a rate of dissociation (k_(off)    rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and    10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as    between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   329. Compound or construct, that comprises or essentially consists    of one or more parts or fragments according to any of aspects 299 to    328, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.-   330. Compound or construct according to aspect 329, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.-   331. Compound or construct according to aspect 329, in which said    one or more linkers, if present, are one or more amino acid    sequences.-   332. Nucleic acid or nucleotide sequence, that encodes a part or    fragment according to any of aspects 299 to 328 or a compound or    construct according to any of aspects 329 to 331 that is such that    it can be obtained by expression of a nucleic acid or nucleotide    sequence encoding the same.-   333. Composition, comprising at least one part or fragment according    to any of aspects 299 to 328, compound or construct according to any    of aspects 329 to 331, or nucleic acid or nucleotide sequence    according to aspect 332.-   334. Derivative of an amino acid sequence according to any of    aspects 1 to 103, or of a Nanobody according to any of aspects 104    to 150.-   335. Derivative according to aspect 334, that can specifically bind    to HER2.-   336. Derivative according to aspect 335, wherein said derivative    competes with Herceptin® for binding to HER2.-   337. Derivative according to any of aspects 335 or 336, wherein said    derivative inhibits and/or blocks binding of Herceptin® to HER2.-   338. Derivative according to any of aspects 335 to 337, wherein said    derivative is directed against the Herceptin® binding site on HER2.-   339. Derivative according to any of aspects 335 to 338, wherein said    derivative specifically binds to domain IV of HER2.-   340. Derivative according to aspect 335, wherein said derivative    competes with Omnitarg for binding to HER2.-   341. Derivative according to any of aspects 335 or 340, wherein said    derivative inhibits and/or blocks binding of Omnitarg to HER2.-   342. Derivative according to any of aspects 340 or 341, wherein said    derivative is directed against the Omnitarg binding site on HER2.-   343. Derivative according to any of aspects 340 to 342, wherein said    derivative specifically binds to domain II of HER2.-   344. Derivative according to any of aspects 335 to 343, wherein said    derivative competes with Herceptin® and Omnitarg for binding to    HER2.-   345. Derivative according to any of aspects 335 to 344, wherein said    derivative inhibits and/or blocks binding of Herceptin® and Omnitarg    to HER2.-   346. Derivative according to any of aspects 335 to 345, wherein said    derivative inhibits and/or blocks tumor cell proliferation.-   347. Derivative according to any of aspects 335 to 346, wherein said    derivative inhibits, downregulates and/or blocks cell signalling.-   348. Derivative according to any of aspects 335 to 347, wherein said    derivative induces apoptosis in tumor cells.-   349. Derivative according to any of aspects 335 to 348, wherein said    derivative inhibits and/or blocks heterodimerization between ERBB    receptors.-   350. Derivative according to any of aspects 335 to 349, wherein said    derivative inhibits and/or blocks ligand activation of an ERbB    hetero-oligomer comprising HER2 and HER3, HER4 or EGFR.-   351. Derivative according to any of aspects 335 to 350, wherein said    derivative inhibits and/or blocks tumor vascularisation.-   352. Derivative according to any of aspects 335 to 351, wherein said    derivative recruits immune effector cells such as macrophages and    monocytes to the tumor.-   353. Derivative according to any of aspects 335 to 352, wherein said    derivative inhibits and/or blocks TNF induced signalling and/or cell    proliferation.-   354. Derivative according to any of aspects 335 to 353, wherein said    derivative down-regulates HER2 levels and/or downregulates    HER2-mediated signalling pathways.-   355. Derivative according to any of aspects 335 to 354, wherein said    derivative inhibits and/or blocks metalloproteinase-mediated HER2    ectodomain shedding.-   356. Derivative according to any of aspects 335 to 355, wherein said    derivative inhibits, downregulates and/or blocks ligand-mediated    ErbB signalling.-   357. Derivative according to any of aspects 335 to 356, wherein said    derivative inhibits and/or blocks HER2 ectodomain cleavage.-   358. Derivative according to any of aspects 335 to 357, wherein said    derivative inhibits and/or blocks Heregulin-mediated activation of    MAPK/Erk1/2.-   359. Derivative according to any of aspects 335 to 358, wherein said    derivative inhibits and/or blocks PI3K/Akt-   360. Derivative according to any of aspects 335 to 359, wherein said    derivative modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Herceptin®.-   361. Derivative according to any of aspects 335 to 360, wherein said    derivative modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Omnitarg.-   362. Derivative according to any of aspects 335 to 361, that can    specifically bind to HER2 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   363. Derivative according to any of aspects 335 to 362, that can    specifically bind to HER2 with a rate of association (k_(on)-rate)    of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³    M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and 10⁷    M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹.-   364. Derivative according to any of aspects 335 to 363, that can    specifically bind to HER2 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ such as between 10⁻⁴ s⁻¹    and 10⁻⁶ s⁻¹.-   365. Derivative of a compound or construct according to any of    aspects 151 to 234.-   366. Derivative according to aspect 365, that can specifically bind    to HER2.-   367. Derivative according to aspect 366, wherein said derivative    competes with Herceptin® for binding to HER2.-   368. Derivative according to any of aspects 366 or 367, wherein said    derivative inhibits and/or blocks binding of Herceptin® to HER2.-   369. Derivative according to any of aspects 366 to 368, wherein said    derivative is directed against the Herceptin® binding site on HER2.-   370. Derivative according to any of aspects 366 to 369, wherein said    derivative specifically binds to domain IV of HER2.-   371. Derivative according to aspect 366, wherein said derivative    competes with Omnitarg for binding to HER2.-   372. Derivative according to any of aspects 366 or 371, wherein said    derivative inhibits and/or blocks binding of Omnitarg to HER2.-   373. Derivative according to any of aspects 371 or 372, wherein said    derivative is directed against the Omnitarg binding site on HER2.-   374. Derivative according to any of aspects 371 to 373, wherein said    derivative specifically binds to domain II of HER2.-   375. Derivative according to any of aspects 366 to 374, wherein said    derivative competes with Herceptin® and Omnitarg for binding to    HER2.-   376. Derivative according to any of aspects 366 to 375, wherein said    derivative inhibits and/or blocks binding of Herceptin® and Omnitarg    to HER2.-   377. Derivative according to any of aspects 366 to 376, wherein said    derivative inhibits and/or blocks tumor cell proliferation.-   378. Derivative according to any of aspects 366 to 377, wherein said    derivative inhibits, downregulates and/or blocks cell signalling.-   379. Derivative according to any of aspects 366 to 378, wherein said    derivative induces apoptosis in tumor cells.-   380. Derivative according to any of aspects 366 to 379, wherein said    derivative inhibits and/or blocks heterodimerization between ERBB    receptors.-   381. Derivative according to any of aspects 366 to 380, wherein said    derivative inhibits and/or blocks ligand activation of an ERbB    hetero-oligomer comprising HER2 and HER3, HER4 or EGFR.-   382. Derivative according to any of aspects 366 to 381, wherein said    derivative inhibits and/or blocks tumor vascularisation.-   383. Derivative according to any of aspects 366 to 382, wherein said    derivative recruits immune effector cells such as macrophages and    monocytes to the tumor.-   384. Derivative according to any of aspects 366 to 383, wherein said    derivative inhibits and/or blocks TNF induced signalling and/or cell    proliferation.-   385. Derivative according to any of aspects 366 to 384, wherein said    derivative down-regulates HER2 levels and/or downregulates    HER2-mediated signalling pathways.-   386. Derivative according to any of aspects 366 to 385, wherein said    derivative inhibits and/or blocks metalloproteinase-mediated HER2    ectodomain shedding.-   387. Derivative according to any of aspects 366 to 386, wherein said    derivative inhibits, downregulates and/or blocks ligand-mediated    ErbB signalling.-   388. Derivative according to any of aspects 366 to 387, wherein said    derivative inhibits and/or blocks HER2 ectodomain cleavage.-   389. Derivative according to any of aspects 366 to 388, wherein said    derivative inhibits and/or blocks Heregulin-mediated activation of    MAPK/Erk1/2.-   390. Derivative according to any of aspects 366 to 389, wherein said    derivative inhibits and/or blocks PI3K/Akt signalling.-   391. Derivative according to any of aspects 366 to 390, wherein said    derivative modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Herceptin®.-   392. Derivative according to any of aspects 366 to 391, wherein said    derivative modulates HER2 or HER2 mediated signalling via the same    mechanism of action as Omnitarg.-   393. Derivative according to any of aspects 366 to 392, that can    specifically bind to HER2 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.-   394. Derivative according to any of aspects 366 to 393, that can    specifically bind to HER2 with a rate of association (k_(on)-rate)    of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹, preferably between 10³    M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably between 10⁴ M⁻¹ s⁻¹ and    10⁷M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹.-   395. Derivative according to any of aspects 366 to 394, that can    specifically bind to HER2 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹.-   396. Derivative according to any of aspects 334 to 395, that has a    serum half-life that is at least 1.5 times, preferably at least 2    times, such as at least 5 times, for example at least 10 times or    more than 20 times, greater than the half-life of the corresponding    amino acid sequence according to any of aspects 1 to 103 per se,    Nanobody according to any of aspects 104 to 150 per se, or compound    or construct according to any of aspects 151 to 234 per se.-   397. Derivative according to any of aspects 334 to 396, that has a    serum half-life that is increased with more than 1 hours, preferably    more than 2 hours, more preferably more than 6 hours, such as more    than 12 hours, or even more than 24, 48 or 72 hours, compared to the    corresponding amino acid sequence according to any of aspects 1 to    103 per se or Nanobody according to any of aspects 104 to 150 per    se, respectively.-   398. Derivative according to any of aspects 334 to 397, that has a    serum half-life in human of at least about 12 hours, preferably at    least 24 hours, more preferably at least 48 hours, even more    preferably at least 72 hours or more; for example, at least 5 days    (such as about 5 to 10 days), preferably at least 9 days (such as    about 9 to 14 days), more preferably at least about 10 days (such as    about 10 to 15 days), or at least about 11 days (such as about 11 to    16 days), more preferably at least about 12 days (such as about 12    to 18 days or more), or more than 14 days (such as about 14 to 19    days).-   399. Derivative according to any of aspects 334 to 398, that is a    pegylated derivative.-   400. Compound or construct, that comprises or essentially consists    of one or more derivatives according to any of aspects 334 to 399,    and optionally further comprises one or more other groups, residues,    moieties or binding units, optionally linked via one or more    linkers.-   401. Compound or construct according to aspect 400, in which said    one or more other groups, residues, moieties or binding units are    amino acid sequences.-   402. Compound or construct according to aspect 400, in which said    one or more linkers, if present, are one or more amino acid    sequences.-   403. Nucleic acid encoding a compound or construct according to any    of aspects 400 to 402 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same.-   404. Composition, comprising at least one derivative to any of    aspects 334 to 399, compound or construct according to any of    aspects 400 to 402, or nucleic acid or nucleotide sequence according    to aspect 403.

FIGURE LEGENDS

FIG. 1: Anti-HER2 humoral immune response induced after immunisation ofLlama glama with HER2-overexpressing SKBR3 cells. The reactivity ofpre-immune (day 0) and immune sera (day 42 and day of PBL1 take) ofanimals 121 and 122 immunized with whole cells was determined by ELISAusing rhErbB2-Fc as antigen (see Example 3.2). A: total IgG response; B:IgG1 isotype response; C: IgG2 isotype response; D: IgG3 isotyperesponse.

FIG. 2: HER2-specific ELISA analysis of periplasmic preparationscontaining myc-tagged Nanobody protein fragments from selected clones.Periplasmic preparations of soluble Nanobody protein fragments wereadded to wells of an ELISA plate, which had been coated with rhErbB2/Fcantigen and had been additionally blocked with PBS+1% casein. Detectionwas performed by a monoclonal anti-myc antibody followed by an alkalinephosphatase-conjugated polyclonal goat anti-mouse antibody. The ELISAwas developed by a PNPP-substrate as described in Example 6. TheOD-values (Y-axis) were measured at 405 nm by an ELISA-reader. Each barrepresents an individual periplasmic extract.

FIG. 3: Flow cytometric analysis of selected clones (2A1, 2A4, 2C3, 2C5,2D3 and 2G4). Nanobody-containing periplasmic extracts were added toErbB2 overexpressing SKBR3 cells. Detection was performed by amonoclonal anti-myc antibody followed by a PE-labeled polyclonalanti-mouse antibody. Nanobodies binding to cells was measured by anincrease in fluorescence intensity as compared to cells that wereincubated with FACS buffer (PBS+10% FBS) followed by monoclonal anti-mycantibody and PE-labeled polyclonal anti-mouse antibody. Fluorescenceintensity is blotted on the X-axis, the number of events on the Y-axis.

FIG. 4: Herceptin® competitive ELISA. An ELISA plate was coated withSKBR3 vesicles (5 μg/ml) and additionally blocked with PBS+1% casein. 2nM Herceptin® was added to the wells, after which periplasmicpreparations of soluble Nanobody protein fragments were added. Detectionof Herceptin® binding to SKBR3 vesicles was performed by an alkalinephosphatase-conjugated AffiniPure Goat Anti-Human IgG, Fc FragmentSpecific (Jackson ImmunoResearch Labs, Suffolk, UK). The ELISA wasdeveloped by a PNPP-substrate as described in Example 6. The OD-values(Y-axis) were measured at 405 nm by an ELISA-reader. Each bar representsan individual periplasmic extract. The OD value corresponding to themaximal signal represents the OD value measured for binding ofHerceptin® without addition of periplasmic extract containingHER-binding Nanobody. The minimal signal represents the backgroundstaining of non-coated wells incubated only with alkalinephosphatase-conjugated AffiniPure Goat Anti-Human IgG, followed bydetection using a PNPP-substrate. Controls 1-5 represent individualperiplasmic extracts containing non-HER2 binding Nanobodies.

FIG. 5: Herceptin®-competitive FMAT. Dilutions of periplasmic extractscontaining HER2 binding Nanobodies were tested for their ability toblock the binding of Herceptin® to HER2-overexpressing SKBR3 cells asdescribed in Example 8. (−) represents the signal obtained for bindingof Alexa647-labeled Herceptin® without addition of periplasmic extract.Addition of periplasmic extract containing a non-HER2 binding Nanobody(irr) had no influence on the binding of Herceptin® to SKBR3 cells.Periplasmic extracts 2A4, 2A5, 2A6, 2B1, 2B2, 2B4, 2B5, 2C1, 2C3, 2D2and 2D3 blocked binding of Herceptin® to HER2 with more than 80%.

FIG. 6: Herceptin®-competitive FMAT analysis. Nanobodies compete withbinding of Herceptin® to SKBR3 cells in a dose-dependent manner asdescribed in Example 8.

FIG. 7: Omnitarg-Fab competitive FMAT. Dilutions of periplasmic extractscontaining HER2 binding Nanobodies were tested for their ability toblock the binding of Omnitarg-Fab (OT-Fab) to HER2-overexpressing SKBR3cells as described in Example 9. (cells) represents the signal obtainedfor binding of biotinylated OT-Fab without addition of periplasmicextract. Addition of periplasmic extract containing a non-HER2 bindingNanobody (irr) had no influence on the binding of OT-Fab to SKBR3 cells.Periplasmic extracts 47A8, 47A11, 47B1, 47B12, 47D1, 47D4, 47D5, 47E7,47F5 and 47G7 blocked binding of OT-Fab to HER2 with more than 85%.

FIG. 8: Growth inhibitory effect of monovalent HER2 binding Nanobodieson ErbB2-overexpressing SKBR3 cells. SKBR3 cells were seeded in 96 wellplates and allowed to adhere as explained in Example 11. HER2-bindingNanobodies 5F7, 2A5, 2A4, 2D3 and 2C3, non-HER2 binding irrelevantNanobody 12B2 or medium alone were added and the cells were incubatedfor 3 days. During the last 24 h, cells were pulsed with 1 μCi[³H]-thymidine. Incorporation of [³H]-thymidine was measured asdescribed in Example 11.

FIG. 9: Herceptin®-competitive FMAT. Dilutions of monovalent, bivalentand bispecific Nanobodies were tested for their ability to block thebinding of Herceptin® to HER2-overexpressing SKBR3 cells as described inExample 12. Bispecific Nanobodies 2A4-9GS-ALB1 and 2A5-9GS-ALB1 blockedthe binding of Herceptin® to HER2-expressing SKBR3 cells to the sameextent as the monovalent 2A4 and 2A5 Nanobodies respectively. Bivalent2A4-9GS-2A4 and 2A5-9GS-2A5 Nanobodies blocked the binding of Herceptin®to HER2-expressing SKBR3 cells to a greater extent than their monovalentformat. A: 2A4 derivatives; B: 2A5 derivatives.

FIG. 10: Design of biparatopic Nanobody expression vector as describedin Example 13.1.

FIG. 11: SKBR3 cell proliferation assay with biparatopic Nanobodiespurified from periplasmic extracts derived from plate 27 by PhyTip200⁺.Biparatopic Nanobodies 27A2-35GS-2D3, 27A5-35GS-2D3, 27B3-35GS-2D3,27B5-35GS-2D3, 27C4-35GS-2D3, 27D3-35GS-2D3 and 27D6-35GS-2D3 blockSKBR3 cell proliferation to a greater extent than 50 nM Herceptin®.Biparatopic Nanobodies 27A7-35GS-2D3, 27A9-35GS-2D3, 27A11-35GS-2D3,27A12-35GS-2D3, 27B11-35GS-2D3, 27C11-35GS-2D3 and 27D7-35GS-2D3 displayan agonistic effect.

FIG. 12: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 anddummy-2D3 biparatopic Nanobodies.

FIG. 13: Sensorgram of monovalent 2D3 and biparatopic Nanobodies27B7-35GS-2D3, 27C3-35GS-2D3 and 27H5-350S-2D3.

FIG. 14: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 andbiparatopic Nanobodies 27A3-35GS-2D3, 27E7-35GS-2D3 and 27D1-35GS-2D3.

FIG. 15: Herceptin®-competitive FMAT. Dilutions of monovalent 2D3,bivalent 2D3-35GS-2D3 and biparatopic Nanobodies combining theHerceptin®-competitive 2D3 and a HER2-binding or dummy Nanobody weretested for their ability to block the binding of Herceptin® toHER2-overexpressing SKBR3 cells as described in Example 14.2. A:Bivalent 2D3-35GS-2D3 and biparatopic Nanobodies 27H3-35GS-2D3 and27D1-35GS-2D3 block binding of Herceptin® to HER2 expressed on SKBR3cells more efficiently than monovalent 2D3 Nanobody. B: Nanobodies 27A3,27A5 and 30D10 have no influence on the Herceptin®-competitive behaviorof Nanobody 2D3 when fused to its N-terminal end, spaced by a 35GSlinker. C: Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody have aninhibitory effect on the Herceptin®-competitive potential of 2D3 whenfused to its N-terminal end, spaced by a 35GS linker.

FIG. 16: Omnitarg-Fab competitive FMAT. Dilutions of OT-Fab, monovalent2D3 and biparatopic Nanobodies 27C3-35GS-2D3, 27A5-35GS-2D3,27H3-35GS-2D3 and dummy-35GS-2D3 were tested for their ability to blockthe binding of OT-Fab to HER2-overexpressing SKBR3 cells as described inExample 14.3. None of the bipartope Nanobodies, nor monovalent 2D3blocked the binding of OT-Fab to HER2 expressed on SKBR3 cells. OT-Fabblocked binding of biotinylated OT-Fab in a dose-dependent manner.

FIG. 17: Effect of biparatopic Nanobodies on SKBR3 tumor cellproliferation. Biparatopic Nanobodies 27A5-35GS-2D3, 27A3-35GS-2D3 and30D10-35GS-2D3 significantly block proliferation of SKBR3 tumor cellsand to a greater extent than the monovalent 2D3 and dummy-2D3biparatopic Nanobody.

FIG. 18: Effect of biparatopic Nanobodies on AKT signaling in SKBR3cells (see Example 16). Biparatopic 27A3-35GS-2D3 and 27A5-35GS-2D3,Herceptin®, but not dummy-2D3 biparatopic or monovalent 2D3 Nanobodyinhibits AKT phosphorylation in whole SKBR3 cell lysates.

FIG. 19: Sensorgram of HER2-ECD binding to 2D3, 47D5 or the biparatopicNanobody 2D3-35GS-47D5.

FIG. 20: Effect of biparatopic Nanobodies combiningHerceptin®-competitive and Omnitarg competitive Nanobodies, monovalentNanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® onHRG-mediated activation of mitogen-activated protein kinase (MAPK).

FIG. 21: Effect of biparatopic Nanobodies combiningHerceptin®-competitive and Omnitarg competitive Nanobodies, monovalentNanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® onHRG-mediated activation of Akt signaling.

FIGS. 22A and 22B: Model of NB-2D3 (blue) linked to another Nanobody(cyan) docked on HER-2 (red). The linker is shown in black. N denotesthe N-terminus of NB-2D3; C is the C-terminus of Nb-2D3.

FIG. 23: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in the biparatopicconstruct 5F7-35GS-47D5 with appropriate linker length. None of theresidues of the linker or at the connection points of the linker withthe Nanobodies (NB-1 and NB-2) have a high energy penalty value.

FIG. 24: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in the biparatopicconstruct 47D5-35GS-5F7 with unappropriate linker length. High energypenalty values are observed at the C-terminal connection of the linkerwith the N-terminal end of the second Nanobody (NB-2).

FIG. 25: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in a biparatopicconstruct with the same Nanobodies as in FIG. 23 but with a longerlinker length (47D5-40GS-5F7). We see that the high energy penaltyvalues at the connection of the C-terminal end of the linker with theN-terminal end of NB-2 are reduced suggesting a more appropriate linkerlength. The energy penalty values at both ends of the linker are stillhigher than those observed in FIG. 22, indicating a still not optimallinker.

FIG. 26A: Backbone RMSD (Å²) between the 5F7-linker-47D5 constructs(built by homology modelling) with the individual Nanobodies 5F7 and47D5 in their unlinked binding mode. The linker length varies from 5 to35.

FIG. 26B: Ribbon view of the 5F7-linker-47D5 biparatopic construct for 2linker lengths. The binding mode of the individual Nanobodies is shownin blue; the biparatopic constructs are in red. The HER-2 target isomitted for clarity. On the left side: a 35GS linker is used between the2 Nanobodies and a very limited deviation from the individual bindingmodes is observed. On the right side: a 5GS linker is used and it canclearly been observed that both Nanobodies in the biparatopic constructsignificantly deviate from their optimal binding mode.

FIGS. 27A-K: Figures illustrating some of the preferred aspects and someof the advantages of the present invention, including the multiparatopicpolypeptides of the invention.

EXAMPLES Example 1 Procurement of the Extracellular Domain of HER2 forUse as Selection Antigen in Phage Display

1.1 Cloning of Extracellular HER2 Domain cDNA was isolated from SKBR3breast cancer cells. The isolation of total RNA and cDNA synthesis wasdone according to standard protocols (Sambrook, Molecular cloning:Laboratory manual, 2^(nd) edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)). The coding sequence of theextracellular domain of the HER2 antigen was amplified by PCR usingprimer For-ErbB2 ECD; GCGAGCACCCAAGTGTGCACC (SEQ ID NO: 2392) and primerRev-ErbB2 ECD: CTGCTCGGCGGGGCAGCCCTT (SEQ ID NO: 2393). The PCRconstruct was then cloned into the pCR4-TOPO cloning vector (Invitrogen,Paisley, UK). Clone 4 having the correct sequence was then amplified byPCR using primers (For-pST ErbB2 ECD:GGCGCGCCGACTACAAAGACGATGACGACAAGAGCACCCAAGTGTGCACC (SEQ ID NO: 2394) andRev-pST ErbB2 ECD: CGGCTCGAGCTATTAATGAGAATGGTGATGGTGCTCGGCGGGGCAGCCCTT(SEQ ID NO: 2395)) that were designed to introduce restriction sites atthe beginning and the end of the fragment encoding the HER2-ECD. The PCRproduct was then cloned via AscI and XhoI into the plasmidpSecTag-HygroA (Invitrogen, Paisley, UK). As such, the coding sequenceof the HER2-ECD was fused in frame with the Ig-κ chain leader sequenceat its N-terminal end followed by a Flag tag and a polyhistidine tag atthe C-terminus. The sequence of different clones was determined bysequencing according to standard protocols.

1.2 Expression of the Extracellular Domain of HER2 in HEK 293 Cells,Purification of the Recombinant Protein

Expression of the extracellular domain of HER2 was performed in HEK293Tcells. HEK293T cells were seeded at 2×10⁶ cells in 20 ml Dulbecco'sModified Eagle's Medium (DMEM) containing 10% FBS in T75 tissue cultureflasks and allowed to adhere overnight. The next day, culturesupernatant was removed and the cells were transiently transfected withpurified pSecTag-HygroA plasmid DNA using Fugene-HD (Roche, Basel,Switzerland) as transfection agent. Cells were grown for an additional72 h in DMEM containing 0.1% FBS, after which the culture supernatantwas collected and filter-sterilized on a 0.22 μm filter (Millipore). Theconstruct was then further purified out of the culture supernatant byimmobilized metal affinity chromatography (IMAC) and size exclusionchromatography (SEC).

Detection of the recombinant protein was performed by ELISA. Maxisorp96-well plate (Nunc, Wiesbaden, Germany) was coated with an anti-flagmonoclonal antibody (Sigma Aldrich, Bornem, Belgium). Unspecific bindingwas blocked with 2% milk powder in PBS for 2 hours. All prior andsubsequent washes were performed with PBS. Afterward, eluate fractionswere incubated for 2 hours at room temperature, followed by incubationwith Herceptin®. Detection of the recombinant HER2-ECD was performedwith a horseradish peroxidase conjugated anti-IgG antibody (JacksonImmunoresearch Laboratories, Suffolk, UK). Development of the ELISA wasperformed with TMB substrate (Pierce, Rockford, Ill.) according to thespecifications of the manufacturer

Example 2 Procurement of Omnitarg-Fab for Use as Competitive Agent inPhage Display and Screening Assays 2.1 Cloning of Omnitarg-Fab

Omnitarg-Fab was constructed by gene assembly. The amino acid sequenceof variable light and variable heavy chain of Omnitarg was derived frompatents WO 2006/044908 and WO 2004/048525. The sequence wasbacktranslated and codon optimized using Leto 1.0 Gene optimizationsoftware (www.entechelon.com). Oligonucleotide primers for assembly ofthe variable light chain (V_(L)), variable heavy chain (V_(H)), constantlight chain (C_(L)) and constant domain 1 of the heavy chain (CH₁) ofthe Omnitarg-Fab were designed (Tables C-5 and C-6) and assembly PCRperformed. The introduced restriction sites SfiI and BsiWI for theV_(L), KpnI and BstEII for the V_(H), BsiWI and AscI for the C_(L), andBstEII and NotI for the CH₁ were utilized for sequential cloning into anin-house expression vector derived from pUC119 which contained the LacZpromoter, a resistance gene for ampicillin or carbenicillin, amulticloning site and the gen3 leader sequence. In frame with theOmnitarg-Fab coding sequence, the vector coded for a C-terminal c-myctag and a (His)6 tag. Oligonucleotide sequences were designed to have a15 nucleotide overlap with 5′ and 3′ overlapping oligonucleotides. Threeconsecutive PCR overlap extension rounds were performed using ExpandHigh fidelity PCR system (Roche, Basel, Switzerland) to obtain V_(L),V_(H), C_(L) and CH₁ respectively. The obtained PCR fragments werecloned into the pCR4-TOPO cloning vector (Invitrogen, Paisley, UK).Plasmid. DNA was prepared from clones having the correct sequence. Thefragments were isolated from the pCR4-TOPO cloning vector viarestriction with the appropriate enzymes and extraction of the fragmentsfrom agarose gel. The fragments were then consecutively cloned into thein-house expression vector.

2.2 Expression of the Omnitarg-Fab in E. coli Cells, Purification of theRecombinant Protein

The Omnitarg-Fab fragment was expressed in E. coli as His6-taggedprotein and subsequently purified from the culture medium by immobilizedmetal affinity chromatography (IMAC) and size exclusion chromatography(SEC).

Omnitarg-Fab was biotinylated using EZ-Link Sulpho-NHS-LC-Biotinlabeling kit according to the manufacturer's instructions (Pierce,Rockford, Ill.). Removal of free biotin was performed on Zeba DesaltSpin columns according to the manufacturer's instructions (Pierce,Rockford, Ill.).

Example 3 Identification of HER2 Binding Nanobodies 3.1 Immunizations

After approval of the Ethical Committee of the Faculty of VeterinaryMedicine (University Ghent, Belgium), 2 llamas (121, 122) wereimmunized, according to standard protocols, with 6 intramuscularinjections at biweekly intervals of SKBR3 human tumor cells which arederived from a breast tumor and contain an amplified HER2 gene andoverexpress HER2 p185 tyrosine kinase (SKBR3; ATCC HTB-30; LGCPromochem, Middlesex, UK). Each dose consisted of approximately 5×10⁷freshly harvested SKBR3 cells.

3.2 Evaluation of Induced Responses in Llama

At day 0, 42 and 81 (time of PBL collection), sera were collected toevaluate the induction of immune responses in the animals against HER2by ELISA. In short, 2 μg/ml recombinant human ErbB2/Fc chimera(rhErb2-Fc; R&D Systems, Minneapolis, Minn.) were immobilized overnightat 4° C. in a 96 well Maxisorp plate (Nuns, Wiesbaden, Germany). Wellswere blocked with a casein solution (1% in PBS). After addition of serumdilutions, specifically bound immunoglobulins were detected using a goatanti-llama horseradish peroxidase conjugate (Bethyl Lab. Inc.,Montgomery, Tex.), showing that for all animals a significant antibodydependent immune response against HER2 was induced (FIG. 1A). Theantibody response was mounted both by the conventional and the heavychain only antibody expressing B-cell repertoires since specificallybound immunoglobulins could be detected with antibodies specificallyrecognizing the conventional llama IgG1 antibodies (FIG. 1B) or theheavy-chain only llama IgG2 (FIG. 1C) and IgG3 (FIG. 1D) antibodies.

3.3 Library Construction

When an appropriate immune response was induced in llama, four daysafter the last antigen injection, a 150 ml blood sample was collectedand peripheral blood lymphocytes (PBLs) were purified by a densitygradient centrifugation on Ficoll-Paque™ (Amersham Biosciences, Uppsala,Sweden) according to the manufacturer's instructions. Next, total RNAwas extracted from these cells and used as starting material for RT-PCRto amplify Nanobody encoding gene fragments. These fragments were clonedinto a phagemid vector derived from pUC119 which contained the LacZpromoter, a coliphage pill protein coding sequence, a resistance genefor ampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the Nanobody® coding sequence, the vector codedfor a C-terminal c-myc tag and a (His)6 tag. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

3.4 Selections

Phage libraries obtained from llamas 121 and 122 were used for differentselections.

In a first selection, ErbB2/Fc chimera (R&D Systems, Minneapolis, Minn.,US) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany)at 20, 5 and 1 nM. Following incubation with the phage libraries andextensive washing, bound phage was aspecifically eluted with trypsin (1mg/ml).

In a second selection, ErbB2/Fc chimera (R&D Systems, Minneapolis, US)was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 20nM. Following incubation with the phage libraries and extensive washing,bound phage was specifically eluted with Herceptin® (Genentech, Roche).

In a third selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After extensive washing, thebiotinylated ErbB2/Fc was captured on a neutravidin coated solid phase.Bound phage was aspecifically eluted with trypsin (1 mg/ml).

In a fourth selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After adding a 100-fold excess ofnon-labeled HER2, the biotinylated ErbB2/Fc was captured on aneutravidin coated solid phase. Bound phage was aspecifically elutedwith trypsin (1 mg/ml).

In a fifth selection, phage libraries were incubated withHerceptin®-captured ErbB2/Fc. After extensive washing, bound phage wasaspecifically eluted with trypsin (1 mg/ml)

In a sixth selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After extensive washing, thebiotinylated ErbB2/Fc was captured on a neutravidin coated solid phase.Bound phage was specifically eluted with Omnitarg-Fab.

In a seventh selection, phage libraries were incubated withHerceptin®-captured ErbB2/Fc. After extensive washing, bound phage wasspecifically eluted with Omnitarg-Fab.

In an eighth selection, phage libraries were incubated with biotinylatedextracellular domain of HER2 captured on a neutravidin coated solidphase. After extensive washing, bound phage was specifically eluted withOmnitarg-Fab.

In a ninth selection, phage libraries were incubated with biotinylatedextracellular domain of HER2 captured on a neutravidin coated solidphase. After extensive washing, bound phage was specifically eluted withHerceptin®.

In all selections, enrichment was observed. The output from eachselection was recloned as a pool into an expression vector derived frompUC119 which contained the LacZ promoter, a resistance gene forampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the Nanobody® coding sequence, the vector codedfor a C-terminal c-myc tag and a (His)6 tag. Colonies were picked andgrown in 96 deep-well plates (1 ml volume) and induced by adding IPTGfor Nanobody expression. Periplasmic extracts (volume: ˜80 μl) wereprepared according to standard methods (see for example the prior artand applications filed by applicant cited herein).

Example 4 Detection and Isolation of HER2-Specific Heavy Chain AntibodyProducing B-Cells

PBMC were isolated from peripheral blood samples from llamas immunizedwith HER2-Fc or SKBR3 human tumor cells using Ficoll density gradientcentrifugation. These were then resuspended in cell culture medium andpartially depleted from monocytes by adherence to the surface of plastictissue culture T-flasks.

Next, non-adherent PBMC were collected from the flasks, washed with FACSbuffer (PBS/10% FCS) at 4° C. and resuspended in the same ice-coldbuffer. These were then stained using a combination of Alexa 488 labeledHER2-Fc (produced in-house, using Invitrogen (Paisley, UK) activatedAlexa 488 and HER2-Fc recombinant protein from R&D Systems (Minneapolis,Minn.)), phycoerythrin labeled mouse-anti-llama IgG2 and -3 monoclonalantibodies (produced in-house, using purified phycoerythrin fromCyanotech, (Kailua-Kona, Hi.) crosslinked using the sulfo-SMCCheterobifunctional linker from Pierce-Endogen (Rochford, Ill.) toin-house produced and purified monoclonal antibodies originallydescribed in Daley et al. (Clin. Diagn. Lab. Immunol. 2005, 12: 380)),Alexa 647 labeled mouse-anti-llama IgG1 monoclonal antibody (producedin-house), Alexa 647 labeled mouse-anti-llama monocyte and neutrophilantibody DH59B (purified antibody obtained from VMRD Inc. (Pullman,Wash.)) and dead cell specific dye TOPRO3 (Invitrogen, Paisley, UK). Insome experiments, in-house Alexa 647 labeled recombinant human IgG1 Fcfragment (R&D Systems, Minneapolis, Minn.) was added to the staincombination as well.

Stained samples were washed thoroughly using cold FACS buffer andanalyzed on a standard two-laser BD FACSAria cell sorter equipped withthe ACDU microtiter plate single-cell deposition option (BD Biosciences,Franklin Lakes, N.J.). During acquisition and analysis, a gate was seton lymphocytes based on their forward/side scatter profile, whichoverlaps considerably with monocytes in llama. Doublet events wereeliminated from acquisition and analysis by forward as well as sidescatter pulse processing, eliminating all events which might beoriginating from more than one cell. Dead cells, monocytes and B-cellsexpressing conventional antibody on their cell membrane were removedfrom further analysis by gating out all remaining events havingfluorescence over background (unstained PBMC) in the Alexa647/TOPRO3channel. In some experiments, Alexa 647 labeled recombinant Fc fragmentwas used to stain the PBMC additionally. In these experiments, B-cellsproducing antibody binding Fc were also rejected from analysis andsorting by similar Alexa 647 channel exclusion, so as to avoid isolationof B-cells binding the Fc region of the fusion protein. In thephycoerythrin channel. B-cells displaying heavy chain antibody on theircell membrane could be clearly differentiated from any other remaininglymphocyte-type cells, and another gate was set on this population.Lastly, antigen-binding heavy chain IgG expressing B-cells cells weredetected as a discrete high fluorescence intensity peak population inthe Alexa 488 channel histogram distinct from the main population beingno more fluorescent in this channel than when no Alexa 488 labeledantigen was added. Individual antigen binding B-cells were collected inseparate wells of 96-well PCR plates in the ACDU, using DiVa softwarepredefined stringent single-cell sorting criteria to avoid anydouble-cell droplet or adjacent-droplet double cell sorting. Typically,only 1-5% of heavy chain B-cells were found to bind antigen.

Example 5 Amplification and Cloning of HER2-Specific Heavy ChainAntibody Variable Regions

Individual B-cells expressing heavy chain antibodies binding HER2-Fc orthe HER2 region of the fusion protein specifically were sorted into96-well plates containing 40 μl of RT-PCR buffer (Superscript IIIOne-step RT-PCR kit, Invitrogen, Paisley, UK) per well, as described inExample 5, and stored at −80° C. For variable region gene sequencerecovery, plates were thawed at room temperature and a mix of NP-40(Roche Applied Sciences, Indianapolis, Ind.), gene specific 5′ and 3′primers and RT-PCR enzyme mix were added to a total volume of 50microliter per well by an automated liquid handler (Tecan, Männedorf,Switzerland). After reverse transcription and first PCR amplification ina standard thermal cycler, a 2 microliter aliquot was removed from allwells and amplified in a nested PCR reaction using a proof-readingthermostable polymerase, or blend of polymerases containing at least oneproof-reading enzyme. The 5′ nested primer contains the nucleotidesequence required for directional TOPO cloning (Invitrogen, Paisley,UK). The 3′ primer is designed to allow for the in-frame fusion ofvariable region gene framework 4 to vector encoded detection (c-myc) andpurification (6His) peptide tags. Amplicons were detected fromindividual wells using ethidium bromide stained agarose gels and/or inmicrotiter plates via PicoGreen DNA binding fluorescent dye assay(Invitrogen, Paisley, UK). Typically, up to 60% of wells contained asingle and sharply defined amplification product, whereas control wellsin the same plate not having received any cells were completely devoidof amplification product.

The amplicons from nested PCR wells containing detectable product werethen ligated into an E. coli expression vector in a homogenous ligationreaction, by mixing an aliquot of unpurified PCR mix with atopoisomerase-activated expression vector (in-house developed IPTGinducible E. coli Nanobody expression vector, adapted to allowdirectional TOPO cloning by Invitrogen's custom services department).The ligation mixture was then pipetted onto electrocompetent E. colicells pre-aliquotted in a 96-well format electroporation chamber array(BTX Products of Harvard Apparatus, Holliston, Mass.), and cells weretransformed by electroporation using a BTX pulse generator.

Transformation mix was spread on selective agarose, multiple individualsubcolonies picked and grown in 96-well deep well plates containingliquid selective medium by a QP Expression colony picker/rearrayersystem (Genetix, New Milton, Hampshire, UK).

Periplasmic extracts (volume: ˜80 μl) were prepared according tostandard methods (see for example the prior art and applications filedby applicant cited herein).

Example 6 Anti-HER2 Nanobodies Recognize Extracellular HER2 Domain

Periplasmic extracts of individual Nanobodies were screened for HER2specificity by ELISA on solid phase coated ErbB2/Fc chimera (R&DSystems, Minneapolis, Minn.). Detection of Nanobody fragments bound toimmobilized recombinant HER2 antigen was carried out using an in housemade mouse anti-myc antibody (2 mg/ml) detected with alkalinephosphatase-conjugated anti-mouse IgG (Sigma Aldrich, Bornem, Belgium).The signal was developed by adding PNPP substrate solution and detectedat a wavelength of 405 nm. FIG. 2 is illustrative of typical ELISAresults, showing a high hit rate of positive clones.

Sequences of different HER2 binding clones are depicted in Tables B-1,B-2 and B-3. Alignment of the different HER2 binding clones based onCDR3 similarity is depicted in Table C-1.

Example 7 Anti-HER2 Nanobodies Recognize Cell Surface Exposed ReceptorEpitopes

To verify whether the Nanobodies are able to recognize cell surfaceexpressed HER2, binding to breast cancer tumor cell line SKBR3 wasassessed by flow cytometry. Cell binding assays were carried out byinitially incubating 200,000 cells with Nanobody-containing periplasmicpreparation obtained in Examples 3 and 5 or relevant controls. Afterincubation, the cells were washed with FACS buffer. Cells weresubsequently incubated successively with an in-house mouse anti-myc-tagmonoclonal antibody and phycoerythrin labeled goat anti-mouse F(ab′)₂fragments (Jackson ImmunoResearch, Suffolk, UK). To omit signals arisingfrom dead cells, a TOPRO-3 (Invitrogen, Paisley, UK) staining wascarried out. Cells were finally analyzed on a BD FACSArray BioanalyzerSystem (BD Biosciences, Franklin Lakes, N.J., US).

FIG. 3 depicts binding of several Nanobody constructs to SKBR3 cells asmeasured by flow cytometric analysis. It can be seen that the constructs2A1, 2A3, 2C3, 2C5, 2D3 and 2G4 show clearly discernable shifts influorescence intensity as compared to the fluorescence intensity forcells incubated only with FACS buffer in the absence of any constructbut with all appropriate detection agents as used for the detection ofNanobody constructs.

Example 8 Screening for Nanobodies that Compete with Herceptin® for HER2Binding

A competition ELISA was performed to screen for Nanobodies that are ableto inhibit the Herceptin® interaction with HER2. In this competitionELISA, the binding of 2 nM Herceptin® to SKBR3 vesicles was evaluated inthe presence of a 1/20 dilution of Nanobody containing periplasmicextract obtained in the second selection described in Example 3. FIG. 4shows an example of this competitive ELISA, identifying several clonesthat compete with binding of Herceptin® to HER2 expressed on SKBR3vesicles.

Periplasmic extracts obtained in the second and ninth selectiondescribed in Example 3 and periplasmic extracts obtained in Example 5,were also screened in a Herceptin®-competitive homogeneous cell-basedassay to evaluate the capacity of the expressed Nanobodies to blockHerceptin® binding to HER2. The FMAT 8200 HTS system (AppliedBiosystems, Foster City, Calif.) assay was performed as follows: SKBR3cells expressing HER2 were grown in tissue culture flasks, collected andwashed with screening buffer (PBS, 10% FCS) and resuspended in screeningbuffer at a concentration of 2.5×10⁵ cells/ml. Alexa 647-labeledHerceptin® was diluted to 62.5 ng/ml in screening buffer. Periplasmicextracts were diluted in screening buffer to obtain final dilutions of4, 10, 40, 100, 200 and 400. To initiate the competitive screen, 10 μllabeled Herceptin®, 10 μl periplasmic dilution and 20 μl of cells wereadded to each well of FMAT system 384-well plates (PE Biosystems, FosterCity, Calif.) The plates were scanned after 2 hours of incubation. Awell was considered positive if it had a count of over 50 events.Screening of the extracts in this Herceptin® competitive homogeneouscell-based assay identified several clones (SEQ ID NOs: 2051-2113) thatcan block the binding of Herceptin® to HER2 with more than 90% (FIG. 5).

Purified Nanobodies were tested for inhibition of binding ofAlexa647-labeled Herceptin® to HER2 expressed on SKBR3 cells. Serialdilutions of purified Nanobody (concentration range: 20 nM-10 pM) wereadded to SKBR3 cells together with 4×10⁻¹⁰M Alexa647-labeled Herceptin®and incubated for 2 hours, after which plates were scanned. Herceptin®was included as reference (MoAb). Results are shown in FIG. 6.Dose-response curves were observed for all Nanobodies with IC₅₀-valuesranging from 40 pM to 200 pM.

Example 9 Screening for Nanobodies that Compete with Omnitarg-Fab forHER2 Binding

Periplasmic extracts obtained in the sixth and seventh selectiondescribed in Example 3, were screened in an Omnitarg-Fab (OT-Fab)competitive homogeneous cell-based assay to evaluate the capacity of theexpressed Nanobodies to block OT-Fab binding to HER2. The FMAT 8200 HTSsystem (Applied Biosystems, Foster City, Calif.) assay was performed asfollows: SKBR3 cells expressing HER2 were grown in tissue cultureflasks, collected and washed with screening buffer (PBS, 10% FCS) andresuspended in screening buffer at a concentration of 2.5×10⁵ cells/ml.Biotinylated OT-Fab was diluted in screening buffer to obtain a finalconcentration of 0.586 nM. The periplasmic extracts were diluted inscreening buffer to obtain final dilutions of 100. To initiate thecompetitive screen, 5 μl labeled OT-Fab, 10 μl periplasmic dilution, 5μl. FMAT Blue dye-labeled streptavidin (100 ng/ml) and 20 μl of cellswere added to each well of FMAT system 384-well plates (PE Biosystems,Foster City, Calif.). The plates were scanned after 2 hours ofincubation. A well was considered positive if it had a count of over 50events. Screening of the extracts in this OT-Fab competitive homogeneouscell-based assay identified clones that can block the binding of OT-Fabto HER2 with more than >90% (FIG. 7). Sequence analysis showed that allclones that blocked binding of OT-Fab HER2 are identical and represent asingle Nanobody (SEQ ID NO: 2114).

Example 10 Screening of Kinetic Off-Rate Constants Via Surface PlasmonResonance (BIAcore)

RhErbB2-Fc was immobilized on a CM5 sensor chip surface docked inBiacore 3000. Approximately 3600RU of rhErbB2-Fc was immobilized.Experiments were performed at 25° C. Periplasmic extracts were diluted10-fold in running buffer (HBS-EP). The samples were injected for 1 minat a flow rate of 45 μl/min over the activated and reference surfaces.Those surfaces were regenerated with a 3 s pulse of glycine-HClμl-11.5+0.1% P20. As an example, the off rate (k_(off)) of differentNanobodies is documented in Table C-2.

Example 11 Anti-HER2 Nanobodies can Block SKBR3 Cell Proliferation

The growth inhibitory characteristics of isolated Nanobodies wereevaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cellswere detached using 0.25% (vol/vol) trypsin and suspended in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum(FCS), glutamine, and penicillin-streptomycin at a density of 1×10⁵cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into 96-wellmicrodilution plates and allowed to adhere. After overnight adherence,cells were washed with serum-free medium and starved for 4 hours in 100μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone ormedium containing Nanobody (final concentration of 50 nM) was added.After 2 days of incubation, cells were pulsed with 1 μCi [³H]-thymidineand incubated for an additional 24 h prior to freezing at −80° C. Cellswere subsequently thawed and embedded on glass fiber membranes using acell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA).After several washings with water, filters were air-dried and countedusing a γ-counter (Perkin Elmer Life Sciences). Nanobody 2A5 inhibitedSKBR3 proliferation by about 18%. Up to 30% or more inhibition wasachieved with Nanobodies 2C3, 2D3, 2A4 and 5F7 (FIG. 8).

Example 12 Generation of Multivalent/Multispecific Nanobody Formats

To potentially increase the biological effect of Nanobody molecules,bivalent constructs were fused head-to-tail using a GGGGSGGGS linker.

Here we describe the construction and characterization of bivalentNanobodies consisting of two identical anti-HER2 molecules all separatedby a 9 (GS) amino acid linker peptide. DNA segments encoding Nanobodies2A4, 2A5, 2C3, 2D3, 5F7 were head-to-tail fused resulting in constructs2A4-9GS-2A4, 2A5-9GS-2A5, 2C₃₋₉GS-2C3, 2D3-9GS-2D3, 5F7-9GS-5F7.Sequences of these bivalent constructs are listed in Table B-4. AllNanobodies were expressed in E. coli and purified according to standardprotocols (see for example the prior art and applications filed byapplicant cited herein).

The different bivalent Nanobody formats were screened in aHerceptin®-competitive homogeneous cell-based assay to evaluate theircapacity to block Herceptin® binding to HER2 compared to theirmonovalent format. Briefly, 10 μl labeled Herceptin® (62.5 ng/ml), 10 μlNanobody dilution and 20 μl of cells (5×10³ cells) were added to eachwell of FMAT system 384-well plates (PE Biosystems, Foster City,Calif.). The plates were scanned after 2 hours of incubation. FIG. 9shows that the bivalent constructs are more efficient in blocking thebinding of Herceptin® to HER2-expressing SKBR3 cells as compared totheir monovalent formats.

To test whether selected Nanobodies have potential as anticancer agentsin an animal model, a strategy to increase the serum half life ispreferred (as for example described in patent application WO 04/041865),since the serum half life of a mono- or bivalent Nanobody (approximately15 or 30 KDa, respectively) is not optimal for this therapeuticindication. Human serum albumin specific Nanobody ALB1 (SEQ ID NO:2391), cross reactive with mouse serum albumin, was chosen. Here wedescribe the construction of bispecific Nanobodies consisting ananti-HER2 Nanobody and ALB1, all separated by a 9 (GS) amino acid linkerpeptide and resulting in constructs 2A4-9GS-ALB1, 2A5-9GS-ALB1,2C3-9GS-ALB1, 2D3-9GS-ALB1 and 5F7-9GS-ALB1. Sequences of thesebispecific constructs are given in Table B-5.

To test whether the HER2-binding Nanobodies as disclosed herein aboveretain their biological activity in a more complicated molecular contextsuch as a bispecific format, Nanobody formats were screened in aHerceptin®-competitive homogeneous cell-based assay to evaluate theircapacity to block Herceptin® binding to HER2 compared to theirmonovalent and bivalent format. Based on the results shown in FIG. 9, itcan be concluded that fusion of a Nanobody with different antigenspecificity to a HER2-binding Nanobody does not affect the potency ofthe latter.

Example 13 Generation of Biparatopic Formats Combining aHerceptin®-Competing Nanobody with a Library of HER2 Binding Nanobodies

The structural requirement for multispecificity is to fuse two or morebinding domains together, with sufficient flexibility to allowsimultaneous binding to different target epitopes. The simplestbispecific is one that binds to two different and non-overlappingepitopes on the same target in such a way that simultaneous binding tothe target is possible. Robert et al (Int. J. Cancer 1995, 28; 62(3):283-90) have described the design of high avidity biparatopic antibodiesdirected against two different epitopes of the carcinoembryonic antigen.Binding of both arms simultaneously without a significant loss ofentropy will endow ‘biparatopic’ antibodies with increased avidity andhence, increased binding affinity to the target. As a result, higherpotency can be obtained as well as enhanced selectivity. In addition,careful selection of the epitopes targeted on the antigen by thebiparatopic antibody or fragment thereof, combined with rational designof linkers to allow maximal flexibility of the two binding domainswithin the biparatopic antibody, may for example result in the blockingof two or more critical interaction sites of the target, leading toimproved potency.

Using genetic fusion, one Herceptin®-competing Nanobody was combinedwith a repertoire of HER2-binding Nanobodies and this mini-repertoirewas screened for biparatopics with improved binding activity and tumorcell growth inhibitory characteristics compared to the monovalentHerceptin®-competing Nanobody.

13.1 Construction of an Expression Vector for Biparatopic Design

For the construction of biparatope Nanobodies, an expression vector wasadapted to contain the Herceptin®-competitive Nanobody 2D3 (which wasshown to block cell proliferation between 20-30% as monovalent format(see Example 11) and which strongly competes with Herceptin® for bindingto HER2-overexpressing SKBR3 cells) to which other Nanobodies withdifferent HER2-binding specificities can be fused, spaced by a linker(FIG. 10). For the design of this vector, a 35 GS linker was used butother linker lengths can also be used to allow flexibility between thetwo building blocks. The 2D3 Nanobody is placed at the C-terminal end ofthe construct to allow SfiI-BstEII cloning of a full selection output.Alternatively, the 2D3 Nanobody can also be placed at the N-terminal endof the construct to allow cloning of a full selection output at theC-terminal end.

13.2 Generation of a Biparatopic Library

A full selection output retrieved from a selection onHerceptin®-captured rhErbB2/Fc followed by trypsin elution (Example3.4), was unidirectionally cloned to the 2D3 Nanobody. Sequence analysisof a selected number of individual colonies derived from the selectionoutput showed a good diversity in the repertoire: 16 Nanobody familieswere identified in 72 sequences. The ligation mix was transformed intoE. coli cells and the transformation mix spread on selective agarose.Multiple individual subcolonies were picked and grown in 96-well deepwell plates containing liquid selective medium by a QP Expression colonypicker/rearrayer system (Genetix, New Milton, Hampshire, UK).Forty-eight individual colonies were sequenced and analyzed. From 32annotated sequenced, eight different Nanobody families were identified.

Periplasmic extracts (volume: ˜80 μl) were prepared according tostandard methods (see for example the prior art and applications filedby applicant cited herein). The biparatopic Nanobodies were purifiedfrom the periplasmic extracts using PhyTip200⁺ columns (Phynexus, SanJose, Calif.) by a Tecan Evo Robotic system (Promega, Madison, US) andanalyzed for their effects on SKBR3 tumor cell proliferation.

13.3 Effect of Biparatopic Nanobodies on SKBR3 Cell Proliferation

The growth inhibitory characteristics of Nanobodies purified fromperiplasmic extracts by PhyTip200⁺ were evaluated using the breast tumorcell line SKBR3. Briefly, SKBR3 cells were detached using 0.25%(vol/vol) trypsin and suspended in DMEM supplemented with 10% fetal calfserum (FCS), glutamine, and penicillin-streptomycin at a density of1×10⁵ cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into96-well microdilution plates and allowed to adhere. After overnightadherence, cells were washed with serum-free medium and starved for 4hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containingmedium alone or 90 ml of 1% FCS containing medium with 10 μl PhyTip200⁺purified periplasmic extract or 50 nM Herceptin® was added. After 2 daysof incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubatedfor an additional 24 h prior to freezing at −80° C. Cells weresubsequently thawed and embedded on glass fiber membranes using a cellharvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). Afterseveral washings with water, filters were air-dried and counted using a₇-counter (Perkin Elmer Life Sciences).

Herceptin® was able to inhibit cell proliferation of SKBR3 up to 50%.Different subclasses of biparatopic Nanobodies were identified: a groupof biparatopic Nanobodies revealed an inhibitory effect on the ErbB2overexpressing cell line SKBR3 to a lower extent than Herceptin®, asecond group of biparatopic Nanobodies increased cell proliferation anda third group of biparatopic Nanobodies was able to inhibit cellproliferation of SKBR3 cells to an equal or greater extent thanHerceptin®. FIG. 11 shows an example of this ‘single hit’ cellproliferation assay.

Example 14 Characterization of Biparatopic Nanobodies

The biparatopic molecules 28F6-35GS-2D3, 28G5-35GS-2D3, 29E9-35GS-2D3,30D10-35GS-2D3, 27A5-35GS-2D3, 31D11-35GS-2D3, 30E10-35GS-2D3,27A3-35GS-2D3, 27B7-35GS-2D3, 27C3-35GS-2D3, 27D1-35GS-2D3,27E4-35GS-2D3, 27E7-35GS-2D3, 27H3-35GS-2D3, 27H4-35GS-2D3,27H5-35GS-2D3 were expressed in E. coli as c-myc, His6-tagged proteinsand subsequently purified from the culture medium by immobilized metalaffinity chromatography (IMAC) and size exclusion chromatography (SEC).A control biparatopic Nanobody consisting of a dummy (i.e. not bindingto HER2) Nanobody genetically fused to the 2D3 Nanobody, spaced by a35GS linker was used as a control.

14.1 Biparatopic Nanobodies Display Improved Binding to HER2 as Comparedto the Monovalent Building Blocks

The off-rate of the biparatopic Nanobodies was determined by surfaceplasmon resonance on a Biacore 3000 instrument. In brief, rhErbB2-Fc wasimmobilized on a CM5 sensor chip surface docked in Biacore 3000.Approximately 3600RU of rhErb B2-Fc was immobilized. Experiments wereperformed at 25° C. Nanobody binding was assessed at variousconcentrations. The samples were injected for 1 min at a flow rate of 45μl/min over the activated and reference surfaces to allow for binding tochip-hound antigen. Next, binding buffer without Nanobody was sent overthe chip at the same flow rate to allow for dissociation of boundNanobody. After 10 min, remaining bound analyte was removed by injectingregeneration solution (Glycine/HCl pH1.5).

The monovalent 2D3 and biparatopic dummy-2D3 Nanobodies had similaroff-rates in the range of 1E-3 l/s, indicating that fusion of a Nanobodyto the N-terminal end of 2D3 does not interfere with binding of thelatter (FIG. 12). The off-rate of bivalent 2D3-35GS-2D3 is in the rangeof 1E-4 l/s, indicating simultaneous binding of the two Nanobodies.

The off-rate of the biparatopic constructs 2B7-350S-2D3, 27C3-35GS-2D3and 27H5-35GS-2D3 are in the range of 1E-3 l/s (FIG. 13). Theseoff-rates and the binding responses indicate binding by the 2D3paratope, but lack of binding by the other paratope, either bynon-specificity for rhErb2 or an extremely much lower affinity forrhErb2 compared to 2D3 or by sterical hindrance of the epitope by the Fcpart or by the altered conformation after the immobilization procedureon the CM5 sensor chip.

Off-rates of the biparatopic constructs 2D3-35GS-2D3, 27D1-35GS-2D3,27A3-35GS-2D3, 27E7-35GS-2D3 are in the range of 1E-4 l/s (FIG. 14).These off-rates indicate simultaneous binding of the 2 paratopes.

14.2 Herceptin®-Competitive Behavior of Biparatopic Nanobodies

Biparatopic Nanobodies were screened in a Herceptin®-competitivehomogeneous cell-based assay to evaluate the capacity of the expressedNanobodies to block Herceptin® binding to HER2. The FMAT 8200 HTS system(Applied Biosystems, Foster City, Calif.) was used as described inExample 8. Bivalent 2D3-35GS-2D3 Nanobody more efficiently blocksbinding of Herceptin® to HER2 as compared to monovalent 2D3 (FIGS. 15Aand B). Likewise, biparatopic Nanobodies 27H3-35GS-2D3 and 27D1-35GS-2D3block binding of Herceptin® to HER2 expressed on SKBR3 cells moreefficiently than monovalent 2D3. Nanobodies 27A3, 27A5 and 30D10 have noinfluence on the Herceptin®-competitive characteristic of Nanobody 2D3when fused to its N-terminal end, spaced by a 35GS linker (FIG. 15B).Finally, Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody have aninhibitory effect on the Herceptin®-competitive potential of 2D3 (FIG.15C).

14.3 Competitive Binding of Biparatopic Nanobodies with Omnitarg-Fab toHER2.

Biparatopic Nanobodies were screened in an Omnitarg-Fab competitivehomogeneous cell-based assay to evaluate the capacity of the expressedNanobodies to block Omnitarg-Fab binding to HER2. The FMAT 8200 HTSsystem (Applied Biosystems, Foster City, Calif.) was used as describedin Example 9. Biparatopic Nanobodies 2D3-35GS-2D3, 27H3-35GS-2D3,27D1-35GS-2D3, 27A3-35GS-2D3, 27A5-35GS-2D3 and 30D10-35GS-2D3 did notefficiently block the binding of biotinylated Omnitarg Fab (FIG. 16).Non-labeled Omnitarg-Fab inhibited binding of biotinylated Omnitarg-Fabin a dose-dependent manner.

Example 15 Biparatopic Nanobodies Comprising a Herceptin®-Competitiveand a HER2-Binding Nanobodies Inhibit SKBR3 Cell Proliferation

The growth inhibitory characteristics of biparatopic Nanobodies wereevaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cellswere detached using 0.25% (vol/vol) trypsin and suspended in DMEMsupplemented with 10% fetal calf serum (FCS), glutamine, andpenicillin-streptomycin at a density of 1×10⁵ cells/ml. Aliquots of 200μl (2×10⁴ cells) were plated into 96-well microdilution plates andallowed to adhere. After overnight adherence, cells were washed withserum-free medium and starved for 4 hours in 100 μl serum-free medium.Then, 100 μl of 1% FCS containing medium alone or 90 μl of 1% FCScontaining medium with serial dilutions of IMAC/SEC purified biparatopicNanobodies, monovalent 2D3 or 50 nM Herceptin® was added. After 2 daysof incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubatedfor an additional 24 h prior to freezing at −80° C. Cells weresubsequently thawed and embedded on glass fiber membranes using a cellharvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). Afterseveral washings with water, filters were air-dried and counted using aγ-counter (Perkin Elmer Life Sciences).

Biparatopic Nanobodies are able to inhibit cell proliferation of SKBR3cells to an equal or greater extent than Herceptin®. FIG. 17 shows anexample of this cell proliferation assay.

Example 16 Biparatopic Nanobodies Comprising a Herceptin®-Competitiveand a HER2-Binding Nanobody Inhibit AKT Signal Transduction in SKBR3Breast Cancer Cells

Upon overexpression, HER2 may be activated by homodimerisation. HER2plays a major regulatory role in the signalling network involved in manycellular processes, including the p21Ras/Mitogen-Activated ProteinKinase (MAPK) and PI3K/AKT pathways. Treatment of HER2 overexpressingSKBR3 cells with Herceptin® results in reduction in HER2 phosphorylationwhich is linked to inhibition of AKT phosphorylation.

To assess the effect of biparatopic Nanobodies on the AKT pathway inSKBR3 cells, cells were plated in 2% serum containing medium in 24-wellculture plates. The next day, medium was refreshed and 50 nM of eitherbiparatopic Nanobody. Herceptin®, monovalent 2D3 Nanobody or mediumalone was added and incubated for 16 h. The reaction was stopped byaspirating the cell medium. Cells were lysed by addition of lysis buffer(20 mM NP40, 20 mM Tris-HCl pH8, 10% glycerol, 2 mM EDTA, 1 mM sodiumorthovanadate, complete protease inhibitor cocktail, 1% PBS). Proteinconcentration in the lysates was measured using BCA protein assay kit(Pierce) according to the manufacturer's indications. Equal amounts ofprotein were run on 10% polyacrylamide gels and electroblotted ontoInvitrolon PVDF membranes (Invitrogen, Paisley, UK). The presence ofphosphorylated AKT was assessed by probing the blots with Phospho-AKT(Ser473) antibody (Cell Signaling, Danvers, Mass.) and total AKT wasdetected using AKT antibody (Cell Signaling). The blots were visualizedusing a chemiluminescent substrate (Perkin Elmer, Wellesley, Mass.,USA).

As shown in FIG. 18, biparatopic Nanobodies 27A5-35GS-2D3 and27A3-35GS-2D3 significantly block AKT activation in SKBR3 cells, whereasdummy-2D3 biparatopic and monovalent 2D3 Nanobody do not have a visibleeffect on AKT signalling.

Example 17 Construction of Biparatopic Nanobodies Combining Herceptin®-and Omnitarg Competitive Nanobodies

For the construction of biparatopics consisting of aHerceptin®-competitive and Omnitarg-competitive Nanobody, the expressionvector described in Example 13.1 was used. Herceptin®-competitiveNanobodies 2D3 and 5F7 were cloned either at the C-terminal orN-terminal end of Omnitarg-competitive Nanobody 47D5, spaced by a 35GSlinker. Biparatopic Nanobodies 2D3-35GS-47D5, 47D5-35GS-2D3,5F7-35GS-47D5 and 47D5-35GS-5F7 were expressed in E. coli as c-myc,His6-tagged proteins and subsequently purified from the culture mediumby immobilized metal affinity chromatography (IMAC) and size exclusionchromatography (SEC). Two control biparatopic Nanobody consisting of adummy Nanobody genetically fused to the 2D3 or 47D5 Nanobody, spaced bya 35GS linker were used as controls.

Example 18 Characterization of Biparatopic Formats Combining Herceptin®-and Omnitarg Competitive Nanobodies 18.1 Biacore Analysis

A kinetic analysis for 2D3, 5F7 and 47D5 was performed on Biacore todetermine the binding affinity to HER2. In addition, the influence of adummy Nanobody fused to the N-terminal end of 2D3 and 47D5 on thebinding characteristics of the latter to HER2, was analyzed. rhErbB2-Fcwas immobilized on a CM5 sensor chip surface docked in T100.Approximately 3600RU of rhErbB2-Fc was immobilized. Experiments wereperformed at 25° C. Different concentrations of Nanobody (100 nM-0.78nM) were made in running buffer (HBS-EP). The samples were injected for1 min at a flow rate of 45 μl/min over the activated and referencesurfaces.

In Table 2 an overview of k_(d)/k_(off), k_(a), and K_(d) values for theNanobodies is shown. Fusion of a Nanobody at the N-terminal end of theNanobodies 2D3 and 47D5 does not significantly alter the bindingcharacteristics of these Nanobodies to HER2.

The binding of the biparatopic 2D3-35GS-47D5 to HER2 was compared to thebinding of the monovalent building blocks 2D3 and 47D5. Hereto,approximately 90 RU of the respective Nanobodies were immobilized anddifferent concentrations (100-1000 nM) HER2-ECD was injected. As shownin Table C-4, the off-rate of the HER2-ECD from the 2D3-47D5 surface was25× lower than the off-rate on each of the 2D3 and 47D5 surfaces,indicating an avidity effect caused by binding of HER2-ECD on both the2D3 and 47D5 Nanobodies simultaneously (FIG. 19).

18.2 Biparatopic Nanobodies Combining Herceptin®- and OmnitargCompetitive Nanobodies Inhibit Heregulin-Mediated HER2-HER3 Signaling

After ligand-binding, the HER receptors become activated by receptordimerization between either two identical receptors (homodimerization)or different receptors of the same family (heterodimerization). Afterreceptor dimerization, activation of the intrinsic protein kinaseactivity and tyrosine autophosphorylation occurs, recruiting andphosphorylating several intracellular substrates involving theRas-Raf-MAPK, the PI3K/Akt, and other signaling pathways that regulatemultiple biological processes including apoptosis and cellularproliferation. The mitogen-activated protein kinases (Erk1/Erk2) are oneof the key endpoints in signal transduction pathways that ultimatelytrigger cancer cells to divide.

The ability of the biparatopic Nanobodies combining Herceptin® andOmnitarg-competitive Nanobodies to inhibit heregulin (HRG) activation ofMAPK-Erk1/Erk2 was assessed in the following way. MCF7 cells(5×10⁴/well) were plated in serum-containing media in 24-well cultureplates. The next day, media were removed and fresh media containing 0.1serum were added to each well. The next day, prior to the assay, themedia were replaced with serum-free medium. Cells were then incubatedfor 30 min with 50 nM of biparatopic Nanobody 2D3-35GS-47D5,47D5-35GS-2D3, 5F7-35GS-47D5 or 47D5-35GS-5F7, monovalent 2D3, 5F7 or47D5, Omnitarg-Fab or Herceptin®. Cells were then treated with 0.2 nMHRG for 15 min. The reaction was stopped by aspirating the cell medium.Cells were lysed by addition of lysis buffer (20 mM NP40, 20 mM Tris-HClpH8, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, completeprotease inhibitor cocktail, 1% PBS). Protein concentration in thelysates was measured using BCA protein assay kit (Pierce) according tothe manufacturer's indications. Equal amounts of protein were run on 10%polyacrylamide gels and electroblotted onto Invitrolon PVDF membranes(Invitrogen, Paisley, UK). The presence of phosphorylated Erk1/Erk2(p44/42 MAPK) was assessed by probing the blots with phosphor-p44/42MAPK (Thr202/Tyr204) antibody (Cell Signaling, Danvers, Mass.) and totalMAPK was detected using p44/42 MAP kinase (137F5) rabbit mAb (CellSignaling). The blots were visualized using a chemiluminescent substrate(Perkin Elmer, Wellesley, Mass., USA).

As shown in FIG. 20, biparatopic Nanobodies 2D3-35GS-47D5 and5F7-35GS-47D5 significantly block HRG-mediated activation of MAPK to agreater extent than Omnitarg-Fab and Herceptin®, Surprisingly, when theOmnitarg-competing Nanobody 47D5 comprised the N-terminal Nanobody inthe biparatopic constructs, i.e. 47D5-35GS-2D3 and 47D5-35GS-5F7, nosignificant reduction in MAPK activation could be observed. MonovalentNanobodies 2D3, 5F7 and 47D5 could not block HRG-mediated MAPKactivation in MCF-7 cells.

These data suggest that the position of the Nanobodies within thebiparatopic Nanobody greatly influences the potency of the molecule. Inaddition, the length of the linker used to genetically fuse 2 Nanobodiesbiparatopic may be critically important to provide maximal flexibilitybetween the 2 Nanobodies to allow tight binding to their respectivebinding epitope on HER2.

Biparatopic Nanobodies 2D3-35GS-47D5 and 5F7-35GS-47D5 were also shownto inhibit heregulin (HRG)-dependent Akt activation (FIG. 21).Activation of the PI3K signal transduction pathway is important for cellsurvival. Complexes formed between HER2 and either HER3 or EGFR caninitiate these pathways in response to HRG. Incubation of MCF7 breastcancer cells with biparatopic Nanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5inhibited HRG-mediated. Akt activation to a greater extent thanOmnitarg-Fab or Herceptin®. These data suggest that the biparatopicNanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5 may inhibit HER2ligand-activation of P13 kinase and that this inhibition may lead toapoptosis.

Example 19 In-Silico Design of Optimal Linker Lengths in BiparatopicNanobody Formats

In-silico design of optimal linker lengths for a biparatopic Nanobodyformat may for example be performed as follows. The 3-dimensional (3D)coordinates of the binding mode of each individual Nanobody to itsrespective epitope on the target are determined, for example from:

-   -   a. a structure of the Nanobody-target complex determined by        X-ray experiments or NMR experiments.    -   b. a docking model of each Nanobody binding on their respective        epitope on the target. Also a number of potential binding modes        of each Nanobody to the target derived from docking studies can        be used. Docking can be done by e.g. ZDock (Chen and Weng 2002,        Proteins 47(3): 281-294; Chen and Weng 2003, Proteins 51(3):        397-408; Chen et al. 2003, Proteins 52(1): 80-87) and refined by        RDock (Li et al. 2003, Proteins 53(3): 693-707) or by other        methods (Fernandez-Recio et al. 2003, Proteins 52(1): 113-117).    -   c. Binding mode of each Nanobody can be extracted from the same        structure or from separate complexes. In the latter case, the        binding modes of each Nanobody on a different epitope on the        same target can be deduced by structural superposition of the        different complexes.

A linker with a given sequence and thus of given length can be modelledbetween the 2 Nanobodies in different ways:

-   -   a. By homology modelling (Sali, and Blundell J. 1993, Mol. Biol.        234: 779-815)        -   i. The sequence of a construct Nanobody1-linker-Nanobody2 or            Nanobody2-linker-Nanobody1 is drawn and stored in a readable            sequence format (e.g. Fasta)        -   ii. The 3-dimensional coordinates of the biparatopic            construct is built by homology modelling by using the            3-dimensional coordinates (from X-ray, NMR or docking            experiments) of the individual binding modes of the            Nanobodies as a template.    -   b. By de-novo design. A linker between the 2 Nanobodies binding        on a different epitope on the same target can be build by        de-novo design (Hu, et al. 2007, Proc. Natl. Acad. Sci. USA        104(45): 17668-17673).    -   c. Several conformations of the linker are sampled and the        lowest state energy conformations (1 or more) can be considered.

As a non-limiting example, the above was performed for a biparatopicconstruct comprising two Nanobodies. The modelling is shown in FIGS. 22Aand 22B, which show a model of Nanobody 2D3 (blue) linked to anotherNanobody (cyan) docked on HER-2 (red). Both figures show that we candock a Nanobody to a target and predict its binding mode to the target.When doing this for several Nanobodies binding on non-overlappingepitopes on the same target, we can design a linker between theNanobodies to create a multivalent Nanobody construct.

The 3-dimensional coordinates of the in-silico generated linker in thebiparatopic construct are evaluated on at least one of the followingcriteria:

-   -   a. Internal energy strain of the linker; possibly compared to a        set of generated linkers of the same sequence in the free state.        At least one but preferentially several energy terms are used        (e.g. Van der Waals energy, electrostatics energy, dihedral        angle deformation energy, etc.). To calculate the energy values        an atom-based force-field (e.g. CHARMM (Brooks et al. 1983, J.        Comp. Chem. 4: 187-217)) or other means of calculating potential        energy (e.g. potentials of mean force (Muegge and Martin        1999, J. Med. Chem. 42: 791)) can be used.    -   b. Internal energy strain on at least one of the residues        (amino-acids) of the linker. For three biparatopic constructs        5F7-35GS-47D5, 47D5-35GS-5F7 and 47D5-40GS-5F7 energy penalty        values were calculated for each residue in the linker as well as        for 10 residues of each Nanobody connected to the linker. Energy        values are shown in FIGS. 22, 23 and 24.    -   c. The root-mean square deviation (RMSD) between the        3-dimensional coordinates of the 2 Nanobodies in the biparatopic        construct and the 2 Nanobodies in their non-linked (monovalent)        binding mode. The higher this value the less likely this linker        is appropriate. FIG. 25 shows the backbone RMSD (Å²) between the        5F7-linker-47D5 constructs (built by homology modelling) with        the individual Nanobodies 5F7 and 47D5 in their unlinked binding        mode. The linker length varies from 5 to 35. FIG. 25A shows that        the RMSD-value is at a minimum value with linker lengths larger        or equal to 15 residues. When shorter linkers are used (e.g.        linker length=5, 10) we see an increased RMSD indicating that        the Nanobodies in the bivalent construct are deviating from        their monovalent binding mode. These in-silico experiments        suggest that biparatopic constructs with linker lengths lower        than 15 residues will have a significant deviation from the        optimal binding mode of the individual Nanobodies to the target.        In FIG. 25B a ribbon view is shown of the 5F7-linker-47D5        biparatopic construct for 2 linker lengths. The binding mode of        the individual Nanobodies is shown in blue; the biparatopic        constructs are in red. When a 35GS linker is used between the 2        Nanobodies and a very limited deviation from the individual        binding modes is observed. However, when a 5GS linker is used,        both Nanobodies in the biparatopic construct significantly        deviate from their optimal binding mode.    -   d. Scores from scoring functions in homology modelling protocols        which are derived based on a combination of experimental data        and in-silico results (Sali & Overington, Protein Science        3(9):1582-1596, 1994).

As can be seen from the above results, the linker in this specificexample should preferably be at least 15 amino acids in length, withlinkers of between 20 and 40 amino acid residues, such as about 25, 30or 35 amino acid residues, being particularly suited.

Also, constructs with different potentially suitable linker lengths (asdetermined by the above in silico analysis) may be prepared and testedfor affinity/avidity, specificity, or potency using suitable bindingassays or in vitro or in vivo potency assays, for example thosementioned in the present specification. In this way, optimal linkerlength may be determined, confirmed or verified.

Example 20 Construction of Multiparatopic Nanobodies for BroaderBiological Activity

Simultaneous binding of 2 adjacent, non-overlapping epitopes by botharms of a biparatopic Nanobody without significant loss of entropyendows biparatopic Nanobodies with increased binding affinity to thetarget and as a result, higher potency can be obtained. The engineeringof Nanobody fragments to obtain an increased potency or broader activityis not limited to the construction of biparatopic Nanobody fragments.Engineering of triparatopic and even tetratopic Nanobodies with carefulselection of the epitopes targeted on the antigen, combined withrational design of linkers to allow maximal flexibility of the bindingdomains within the multiparatopic antibody, may for example result inthe blocking of several critical interaction sites of the target,leading to improved potency and even an unparalleled biologicalactivity.

Tables

TABLE B-1 Preferred Nanobodies against HER2 obtained as described inExample 3 <Name, SEQ ID #; PRT (protein); -> <13D11, SEQ ID NO: 2051;PRT; -> EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS<2B4, SEQ ID NO: 2052; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS<2G2, SEQ ID NO: 2053; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS<13D2, SEQ ID NO: 2054; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKIVPTDRGGHGTQVTVSS<2D5, SEQ ID NO: 2055; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS<2F4, SEQ ID NO: 2056; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRRGHGTQVTVSS<2C3, SEQ ID NO: 2057; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS<17E3, SEQ ID NO: 2058; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS<17H3, SEQ ID NO: 2059; PRT; -> EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS<17D2, SEQ ID NO: 2060; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGSHGTQVTVSS<2F1, SEQ ID NO: 2061; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKELEWISSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS<2E2, SEQ ID NO: 2062; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<2C2, SEQ ID NO: 2063; PRT; -> EVQLVESGGGLVQGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS<2E3, SEQ ID NO: 2064; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS<13B10, SEQ ID NO: 2065; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS<2D1, SEQ ID NO: 2066; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKILPTNRGSHGTQVTVSS<2H3, SEQ ID NO: 2067; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<2H1, SEQ ID NO: 2068; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVRGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<2C1, SEQ ID NO: 2069; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<15C5, SEQ ID NO: 2070; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<2B3, SEQ ID NO: 2071; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<29H2, SEQ ID NO: 2072; PRT; -> EVQLVESGGGLVQGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<17E4, SEQ ID NO: 2073; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS<17A2, SEQ ID NO: 2074; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKVWPTDRGTHGTQVTVSS<15D1, SEQ ID NO: 2075; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS<17B8, SEQ ID NO: 2076; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS<15C11, SEQ ID NO: 2077; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS<15G8, SEQ ID NO: 2078; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWNGTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKILPAERRGHGTVTVSS<17H4, SEQ ID NO: 2079; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLINYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS<27G8, SEQ ID NO: 2080; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS<38C6, SEQ ID NO: 2081; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQWKIRPTIPMGHGTQVTVSS<2A4, SEQ ID NO: 2082; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS<15G7, SEQ ID NO: 2083; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGTHRNYADSVKGRFTISRDNNKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS<15B7, SEQ ID NO: 2084; PRT; -> EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKSQGYWGQGTQVTVSS<5G4, SEQ ID NO: 2085; PRT; -> EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRFTISRDNAKKTLYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS<13B2, SEQ ID NO: 2086; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHKDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<2E5, SEQ ID NO: 2087; PRT; ->EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<15G1, SEQ ID NO: 2088; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27B1, SEQ ID NO: 2089; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<17E7, SEQ ID NO: 2090; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<17D8, SEQ ID NO: 2091; PRT; ->EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<5F8, SEQ ID NO: 2092; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYALSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<2D4, SEQ ID NO: 2093; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS<13D8, SEQ ID NO: 2094; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<17G8, SEQ ID NO: 2095; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<2H4, SEQ ID NO: 2096; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<2F3, SEQ ID NO: 2097; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS<2F5, SEQ ID NO: 2098; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLOMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<30E10, SEQ ID NO: 2099; PRT; ->KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<29H1, SEQ ID NO: 2100; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<17E2, SEQ ID NO: 2101; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<2B1, SEQ ID NO: 2102; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS<2A5, SEQ ID NO: 2103; PRT; ->EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<13C12, SEQ ID NO: 2104; PRT; ->EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQTVSS<17E10, SEQ ID NO: 2105; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDCTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<27D4, SEQ ID NO: 2106; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQTVSS<15F9, SEQ ID NO: 2107; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<30H9, SEQ ID NO: 2108; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<39C1, SEQ ID NO: 2109; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS<27G2, SEQ ID NO: 2110; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS<2D3, SEQ ID NO: 2111; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<5F7, SEQ ID NO: 2112; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS<47D5, SEQ ID NO: 2114; PRT; ->KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS<14B11, SEQ ID NO: 2115; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS<14B10, SEQ ID NO: 2116; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS<14B4, SEQ ID NO: 2117; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS<14C11, SEQ ID NO: 2118; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS<14B5, SEQ ID NO: 2119; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS<14C6, SEQ ID NO: 2120; PRT; ->EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS<14A4, SEQ ID NO: 2121; PRT; ->EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS<14B3, SEQ ID NO: 2122; PRT; ->EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS<14C1, SEQ ID NO: 2123; PRT; ->EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS<14A12, SEQ ID NO: 2124; PRT; ->EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS<14A2, SEQ ID NO: 2125; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS<14A1, SEQ ID NO: 2126; PRT; ->EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS<17C3, SEQ ID NO: 2127; PRT; ->EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS<46D3, SEQ ID NO: 2128; PRT; ->KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRFTVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS<27H5, SEQ ID NO: 2129; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTOVTVSS<17C2, SEQ ID NO: 2130; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQGTQVTVSS<17D11, SEQ ID NO: 2131; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREFVATISRGGSATYYADSLKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSSLYTSSNVFEYDYWGQGTQVTVSS<15A6, SEQ ID NO: 2132; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS<17B6, SEQ ID NO: 2133; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS<17C5, SEQ ID NO: 2134; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS<15E11, SEQ ID NO: 2135; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDWVATISARGMPAYEDSVKGRFTVSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS<15C2, SEQ ID NO: 2136; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS <2A3,SEQ ID NO: 2137; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDWVATIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIWGQGTQVTVSS <27A5,SEQ ID NO: 2138; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS <2C5,SEQ ID NO: 2139; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDWVATIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS <27G5,SEQ ID NO: 2140; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS<13A9, SEQ ID NO: 2141; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS<29E9, SEQ ID NO: 2142; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS<15D8, SEQ ID NO: 2143; PRT; ->EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS<15G4, SEQ ID NO: 2144; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS<15D12, SEQ ID NO: 2145; PRT; ->EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQRELVATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS<15E12, SEQ ID NO: 2146; PRT; ->EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS<13D7, SEQ ID NO: 2147; PRT; ->EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS<13A8, SEQ ID NO: 2148; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS<15A4, SEQ ID NO: 2149; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS<17F7, SEQ ID NO: 2150; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIAQSIRVMAWYRQPPGKQRDWVGTISSDGTANYADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS<15C8, SEQ ID NO: 2151; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<17A10, SEQ ID NO: 2152; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<27D3, SEQ ID NO: 2153; PRT; ->EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS<13B12, SEQ ID NO: 2154; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATIGSDGTTIYADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<15B2, SEQ ID NO: 2155; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS<15B11, SEQ ID NO: 2156; PRT; ->EVQLVESGGGSVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS<13C9, SEQ ID NO: 2157; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPSIHAMAWYRQAPGKQRDWGATTYSRGGTTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<17D5, SEQ ID NO: 2158; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGIIGTIRTMAWYRQAPGKQRDWVASIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<27B5, SEQ ID NO: 2159; PRT; ->EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<27C7, SEQ ID NO: 2160; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS<13D4, SEQ ID NO: 2161; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS<15G5, SEQ ID NO: 2162; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS<13C4, SEQ ID NO: 2163; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS<46G1, SEQ ID NO: 2164; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKERECVASLYLNGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS<46E4, SEQ ID NO: 2165; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAVGWFRQAPGKERECVASMYLDGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSEYNYWGQGTQVTVSS<17B5, SEQ ID NO: 2166; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREFVASITKFGSTNYADSVKGRFTISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS<15C9, SEQ ID NO: 2167; PRT; ->EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAMAWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDLWGQGTQVTVSS<13D10, SEQ ID NO: 2168; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASSVITLDSNAIGWFRQAPGKEREEVSCIASSDGSTYYAESVKGRFTISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS<17C6, SEQ ID NO: 2169; PRT; ->EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIMAWYRQAPEKQRELVASVSGGGNSDYASSVKGRFTISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYYMPFWGQGTQVTVSS<15A2, SEQ ID NO: 2170; PRT; ->EVQLVESGGGLAQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS<17A8, SEQ ID NO: 2171; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS<15G10, SEQ ID NO: 2172; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS<27A3, SEQ ID NO: 2173; PRT; ->EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS<17H10, SEQ ID NO: 2174; PRT; ->EVQLVESGGGLVQAGGSLSLSCSASGRFFSTRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS<30D10, SEQ ID NO: 2175; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS<15H4, SEQ ID NO: 2176; PRT; ->EVQLVESGGGLVQAGGSLTLSCAPSESTVSFNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS<17B7, SEQ ID NO: 2177; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTMAWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYWGQGTQVTVSS<15D2, SEQ ID NO: 2178; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGVFGPIRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS<17G5, SEQ ID NO: 2179; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS<15B6, SEQ ID NO: 2180; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMAWYRQAPGNQRDWVATIGSAGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS <27F2,SEQ ID NO: 2181; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS<17F5, SEQ ID NO: 2182; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS <17B2,SEQ ID NO: 2183; PRT; -> EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS<27H4, SEQ ID NO: 2184; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS<13A4, SEQ ID NO: 2185; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS<2A1, SEQ ID NO: 2186; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFTISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS<15E10, SEQ ID NO: 2187; PRT; ->EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKERELVAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS<27E7, SEQ ID NO: 2188; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS<47E5, SEQ ID NO: 2189; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS<2G4, SEQ ID NO: 2190; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFTISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS<14D4, SEQ ID NO: 2191; PRT; ->EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS<17A5, SEQ ID NO: 2192; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS<15D10, SEQ ID NO: 2193; PRT; ->EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS<13C2, SEQ ID NO: 2194; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS<17G11, SEQ ID NO: 2195; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS<17A3, SEQ ID NO: 2196; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTISRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS<27B7, SEQ ID NO: 2197; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS<17A6, SEQ ID NO: 2198; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS<17D7, SEQ ID NO: 2199; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS<46D4, SEQ ID NO: 2200; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRFTISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS<27B3, SEQ ID NO: 2201; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS<27E5, SEQ ID NO: 2202; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS<27D6, SEQ ID NO: 2203; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS<30D10, SEQ ID NO: 2204; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS<47G11, SEQ ID NO: 2205; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVAAIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS<27C3, SEQ ID NO: 2206; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE B-2 Preferred Nanobodies against HER2 obtained as described inExample 4 <Name, SEQ ID #; PRT (protein); -> <11A101/1-120, SEQ ID NO:2207; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFNAMGWFRQAPGKEREFVAAISRSPGVTYYADSVKGRFTTSRDNAKNTVYLQMNDLKPEDTAVYYCAADFYLATLAHEYDYWGQGTQVTVSS<11A22/1-122, SEQ ID NO: 2208; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS<12D44/1-122, SEQ ID NO: 2209; PRT; ->KVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRFTISRANAKNTVYLQMNGLKPEDTAVYYCAADFYVSTLAHEYDYWGQSTQVTVSS<12E11/1-122, SEQ ID NO: 2210; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVGGIRWSDGSTYYADSVKGRFTISRDNAKITVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS<13G111/1-123, SEQ ID NO: 2211; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERAFVAAIRWSGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTFTLSTLSHEYDYWGQGTQVTVSS<13F71/1-123, SEQ ID NO: 2212; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTFSNYALAWFRQAPGKEREFVAAINWRSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLIVATLPGEYDYWGQGTQVTVSS<14H61/1-122, SEQ ID NO: 2213; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAAVRWSDDYTYYADSVKGRFTISRDNAKNTVYLQMNSLSPEDTAVYYCAADEILATLPHEYDYWGQGTQVTVSS<22B12/1-124, SEQ ID NO: 2214; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVAGINKSGGITHSADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADAYTVIATLPHEYDYWGQGTQTVSS<14H71/1-123, SEQ ID NO: 2215; PRT; ->EVQLVESGGGLVQAGGSLRLSCEASGLTISSLTMAWFRQAPGKEREFVANIKWSGDRIVYADSVKGRFTISRDSAKNAVNLQMELVESDDTAVYYCAAKHSTVAGLTHEYDYWGQGTQVTVSS<12D51/1-120, SEQ ID NO: 2216; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSAFSIKSMGWYRQAPGKQRELAAVIISSGTTTYADSVKGRFTISRDSAKNTVYLQMDSLKPEDTAVYVCNAVYVSTWGNGYDYWGQGTQVTVSS<11A111/1-126, SEQ ID NO: 2217; PRT; ->EVQLVESGGGLVQAGGSLGLSCAAAGRTFSSSLMGWFRQAPGKEREFVAAITDNGGSTYYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAIYYCAARRSGYYSLSTSPHQYAYWGQGTQVTVSS<13G71/1-124, SEQ ID NO: 2218; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAMGWFRQAPGKERDFVAAITSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQVTVSS<13G74/1-125, SEQ ID NO: 2219; PRT; ->EVQLVESGGGLVQAGGSLRLSCATSGRTFSTYASMGWFRQTPGKEREFVAAITSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQVTVSS<11A71A/1-116, SEQ ID NO: 2220; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGNIDGIITMGWYRQRPGKPREWVGTINSGGDTNYAGSVKGRFTIARDDAKNTMYLQMNGMKPEDTAVYYCKMNRAGIYEYWGQGTQVTVSS<22B101/1-123, SEQ ID NO: 2221; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGPTFSDYAIGWFRQAPGKEREFVAAISSSGISTIYGDSVKGRFDISRDNAKNTVYLQMNRLKPEDTAVYYCAARLFMATPNQGQYYYWGQGTQTVSS<11B42/1-123, SEQ ID NO: 2222; PRT; ->EVQLVESGGGLVQAGDSLRLSCAARSGFTFSNHIMGWFRQAPGKERELIAEITWNGGSTYYADSVKGRFAISRDNALNTVYLQMNSLKPEDTAVYYCAARPSYSTNNVKSYRYWGQGTQVTVSS<13E111/1-124, SEQ ID NO: 2223; PRT; ->EVQLVESGGGLVQAGSSLRLSCALSGRTFSDYAIGWFRQAPGKEREFVAAISGWSGGTTNYADSVKGRFTISRDNGKNTVDLRMNSLKPEDTAVYYCAARPAVVHTRKESYPYWGQGTQVTVSS<14H12/1-125, SEQ ID NO: 2224; PRT; ->EVQLVESGGGLVQAGGSLRLSCIASERTFSSAGVGWFRQAPGKERDFVAAISWNGVTIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARINYSVLTTTSSSYHYWGQGTQVTVSS<13G101/1-123, SEQ ID NO: 2225; PRT; ->EVQLVESGGGLVQPGDSLRLSCSASEGTLSRSRVAWFRQAPGKEREFVTVISGVGTSYADSVKGRFTISRDDAKNTVYLQMNSLKAEDTAIYYCAADFRSTWLSSSGSSYTYWGQGTQVTVSS<13G41/1-121, SEQ ID NO: 2226; PRT; ->EVQLVESGGGLVQPGGSLTLSCVGSGRRFSADVMGWYRQAPGKQREFVASISSGSAINYADSVKGRFTVSRDNAQNTVYLQMNSLKIEDTGVYYCNARRIVNVEGAYRDYWGQGTQVTVSS<22B910/1-121, SEQ ID NO: 2227; PRT; ->EVQLVESGGGLVQPGGSLPLSCAASGSIFRMNDMGWYRQAPGKQRERVATLTSAGNTNYADSVKGRFTISGDDARNTVYLQMNSLNPEDTAVYYCNAKVVVAVEGAKYDYWGQGTQVTVSS<21A81/1-122, SEQ ID NO: 2228; PRT; ->EVQLVESGGGLAQAGGSLRLSCAVFGRSRYGMAWFRRAPGKEREFVAGIAWNGASIGSADSVRGRFTISRDNSENTVYFEMGSLKPEDTAVYYCAICRISWCAGAESDYGYWGQGTQVTVSS<21A92/1-127, SEQ ID NO: 2229; PRT; ->EVQLVESGGGQVQAGGSLRLSCTESGRAFNTRAMGWFRQAPEKEREFVAGITMSGFNTRYADSVKGRFTISRDNAKGTVYLQMSSLKPEDTAVYYCAADSITDRRSVAVAHTSYYYWGQGTQVTVSS<22C712/1-123, SEQ ID NO: 2230; PRT; ->EVQLVESGGGLVQAGGSLGLSCAASGRTFSNYAMGWFRQAPGKEREFVAGISWSGGHTFYADSVKGRFTISRDNTKNTVYLQMNSMRPEDTAVYYCAARLSSVAVASTRYDYWGQGTQVTVSS<11A13/1-125, SEQ ID NO: 2231; PRT; ->EVQLVESGGGLVQAGDSLRLSCVASGGTFGSYAMGWFRQAPGKEREFVATIDWSGDTAFYADSVKGRFTISRDIANDVVYLQMNSLEPEDTAVYYCARNRQSGVASENLRLYTYWGQGTQVTVSS<13G93/1-123, SEQ ID NO: 2232; PRT; ->EVQLVESGGGLAQAGDSLRLSCVDSGSSFSAYAMGWFRQAPGKEREFVAAVSWDGRNTYYADSVKGRFTISRDNAKNTLYLQTTSLRPEDTGVYYCAEDKQSGVSVNPKYAYWGQGTQVTVSS<12C52/1-118, SEQ ID NO: 2233; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGGTFESDTMAWFRQAPGKEREFVARVSWIRTTYYSDSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCAAQTLGRSLYDYWGQGTQVTVSS<12C61/1-126, SEQ ID NO: 2234; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAMAWFRQAPGNERELVSAIGWSGASTYYIDSVEGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASRYSGGVATARRSEYHYWGQGTQVTVSS<21A61/1-125, SEQ ID NO: 2235; PRT; ->EVQLVESGGGLVQAGDSLRLSCVASGDSFNTYTMGWFRQAPGKEREFVAAIRWSGGTTFYGDSVKGRFTISRDYAKNTWYLQMNTLKPEDTAAYYCAAVATYSRNVGSVRNYDYWGQGTQVTVSS<11A121/1-126, SEQ ID NO: 2236; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSEGTFSSYSMGWFRQAPGKDREFVSAITWNGTRTYYRDSVKGRFTISRDNAKNTVQLQMNSLKPEDTAVYYCAVSQPLNYYTYYDARRYDYWGQGTQVTVSS<11A91/1-124, SEQ ID NO: 2237; PRT; ->EAQLVESGGGLVQAGGSLRLSCTASGRTYSTIMGWFRQAPGKEREFVAAIRWSGGSAFYADSVKGRFTISRDNAKNTVYLQMTSLMPEDTAVYYCADTPVYYQRYYDQNAYDYWGQGTQVTVSS<13G72/1-118, SEQ ID NO: 2238; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAMGWFRQAPGKERDFVAAITSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKYYSYYAYDYWGQGTQVTVSS<13E81/1-124, SEQ ID NO: 2239; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSVYHMAWFRQAPGKEREFVAAIRSSGGLFYALSVSGRFTISRDNAKDTMYLQMNVLKPEDTAVYYCAASPVYYIDYSSQYKYGYWGQGTQVTVSS<11B31/1-124, SEQ ID NO: 2240; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGAFGVYHMGWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCATQIYYRTNYYSQNAYDYWGQGTQVTVSS<13G81/1-124, SEQ ID NO: 2241; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFVAVIRSGGTTLYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYLCAAQIYYRTNYYSQNNYDYWGQGTQVTVSS<21A53/1-124, SEQ ID NO: 2242; PRT; ->EVQLVESGGGLVQAGGSLELSCAASGGAFGVYHMGWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRVTISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNVYDYWGQGTQVTVSS<14H51/1-124, SEQ ID NO: 2243; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYTMAWFRQAPGKEREFVAAIRSGATTLYEDSVKGRFTISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNEYDYWGQGTQVTVSS<21A21/1-124, SEQ ID NO: 2244; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHMGWFRQAPGTEREFVAVIRSGGTTLYEDSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYSSQSNYDYWGQGTQVTVSS<21A111/1-124, SEQ ID NO: 2245; PRT; ->EVQLVESGGGLVQAGGSLKLSCAVSGRTIVPYTMAWFRQAPGKEREFVAVTRSGGTTFYADSAKGRFTIARDDAKNTVYLQMNSLKPEDTAVYYCALATAYRTNYSSRDKYDYWGQGTQVTVSS<22B1212/1-122, SEQ ID NO: 2246; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINSGGGSTSYADSVNGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKYLSFYSDYEVYDYWGQGTQVTVSS<11A31/1-120, SEQ ID NO: 2247; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSSGVMAWFRQSPGEEREFLALITRNGETKKTADSVKGRFTISRDNAKNGVSLQMDSLKAEDTAVYYCASDPTYGSGRWTYWGQGTQVTVSS<13E51/1-128, SEQ ID NO: 2248; PRT; ->EVQLVESGGGLNQAGGSLRLSCAASRHTFSGYAMGWFRQAPGKEREFVAAIRWSGGITYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTALYYCARSVTYYSGSHAYTQEGGYARWGQGTQVTVSS<12D121/1-126, SEQ ID NO: 2249; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGRAFSTYGMGWFRQAPGKAREFVAAISRSGTGTYYAGSMKGRFTISRDDAKNTVYLOMNSLKPEDTAVYYCAARQPYASGSHYSSTQYTYWGQGTQVTVSS<13F121/1-119, SEQ ID NO: 2250; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRSFNDYTMGWFRQTPGKEREFVARVWWNGGSAYYADSVKGRFTISIDNAKNTVYLQMNNLTPEDTAVYYCAALYRGRSVYDDWGQGTQVTVSS<13G121/1-127, SEQ ID NO: 2251; PRT; ->EVQLVESGGGLVRAGTSLRLSCADSARTFSSAAMGWFRQAPGKEREFVSAISPIGSSKYYADSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAASSYGSTYYSQGRAYYYDYWGQGTGVTVSS<22B41/1-124, SEQ ID NO: 2252; PRT; ->EVQLVESGGGLVQPGGSLRLSCTVFGRTFSGDVIGWFRQAPGKEREFVAAISTSGGGTDSADSVKGRFTISKENAKNTVYLQMTILKPEDTAVYYCASSPYGPLYRSTHYYDYWGQGTQVTVSS<12D71/1-125, SEQ ID NO: 2253; PRT; ->EVQLVESGGGLVQAGGSLGLSCAASGRTVSTMGWFRQAPGKEREFVTAITWSGDSTNFADSVKGRFTISRDSAKDTVYLQMNNLKPEDTAVYYCAATTYYSGSYISTLSTSYNYWGQGTQVPVSS<13F42/1-111, SEQ ID NO: 2254; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKGRESVATIFVGGTTYYSDSVKGRFTISRDNAKNAVNLQMSNLKPEDTALHYCTICSYRGQGTQVTVSS<12C101/1-111, SEQ ID NO: 2255; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKERESVATIFVGGTTYYSDSVKGRFTISRDNARNAVNPQMNNLKPEDTAVYYCTIGSYRGQGTQVTVSS<14H91/1-127, SEQ ID NO: 2256; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRDVMGWFRQAPGKEREFVAAKTWSGASTYYADSVRGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAARDSSTLDSTYYVGGSYNYWGRGTQVTVSS<13F41/1-111, SEQ ID NO: 2257; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGVGWFRQAPGKERESVATIFVGGTTYYSDSVKGRFTISRDNAKNAVNLQMSNLKPEDTALYYCTIGSYRGQGTQVTVSS<14H21/1-125, SEQ ID NO: 2258; PRT; ->EVQLVESGGGLVQGGSLRLSCVRSGGYFGSYHIGWFRQAPGNEREFVAAITWNGASTSYADSVKGRFTISRSIAENTVYLQMNKVKPEDTAVYYCAARMYGSDWLPRPEDFDSWGQGTQVTVSS<22B610/1-120, SEQ ID NO: 2259; PRT; ->EVQLVESOGGLVQPGGSLRLSCAASGSIFSINAMGWYRPAPOKQRELVARITSTGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVSPSYGSRWYGWGQGTQVTVSS<12C32/1-127, SEQ ID NO: 2260; PRT; ->EMQLVESGGGLVQAGGSLRLSCATSERTFSTYTMAWFRQAPGKEREFVVAIKSSDNSTSYRDSVKGRFTISRDNAKSTMYLQMNSLKPEDTAVYYCAARREYSTIYTARYPGEYVYWGQGTQVTVSS<12D61/1-116, SEQ ID NO: 2261; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRSIFSPNVVGWYRQAPGKQRELVAAVTSGGITNYADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCNARERGIYDSWGQGTQVTVSS<13G31/1-125, SEQ ID NO: 2262; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYKMGWFRQAPGKEREFVAASRWSGGIKYHADSVKGRFTISRDDAKNSIYLQMNTLKPEDTAVYYCAADDYLGGDNWYLGPYDSWGQGTQVTVSS<22C65/1-124, SEQ ID NO: 2263; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGFLFDSYAMGWFRQAPGKEREFVAAIRWSGSATDYSDSVKGRFTISRDNAKNTVYLQMNSLIPEDTAVYYCAARKTYRSLTYYGEYDSWGQGTQVTVSS<11A71/1-125, SEQ ID NO: 2264; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRSIRSVSVMGWYRLAPGNQRELVATITADGITNYADSVKGRFTVSRDNGRNTVYLQMNSLKPEDTAVYYCNVDRLLYYSSGYYQTSVDVWGQGTQVTVSS<11B91/1-125, SEQ ID NO: 2265; PRT; ->EVQLVESGGALVQPGGSLRLSCAASGSIRSINTMGWYRQAPGNQREFVAAVTEGGTTSYAASVKGRFTISRDKAKNTVLLQMDSLKPEDTAVYYCNADRFLYYSAGRYDTGSDIWGQGTQVTVSS<11A81/1-125, SEQ ID NO: 2266; PRT; ->EVQLVESGGALVQPGGSLRLSCAASDSIRSINIMGWYRQAPGKQREFVAAVTEDGSINYAESVKGRFTISRDKAKNALYLQMNSLKPEDMAVYYCNADRVLYYSDSRYYTGSNYWGQGTQVTVSS<11B121/1-127, SEQ ID NO: 2267; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSSASINTMGWYRQAPGEQRELVAEITEGGIINYTDSVKGRFTISRDNAKNTVYLEMNNLKPEDTAVYYCNADRALYRNYSDGRYYTGYDYWGQGTQVTVSS<12D31/1-115, SEQ ID NO: 2268; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRNIFDFNDMGWYRQGPGKEREFVALINVGGVAKYEDSVKGRFTISRDNAENTVYLQMNNLKPEDMAVYYCNARILSRNYWGQGNQVTVSS<11B51/1-127, SEQ ID NO: 2269; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSGRGMGWFRQAPGKEREFVAAVSWSGGNTYYADSVKGRFTISRDNAKSTVYLQMDSLKPEDTAVYYCAASRRFYSGLYYYTDDAYEYWGQGTQVTVSS<13G51/1-127, SEQ ID NO: 2270; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGGTFNGRAVGWFRQAPGEEREFVTGISWSGGSTDYADSVKGRFTISRDNSKNTVSLQMNSLKPEDTAVYYCAASRRFYSGLVYYSVDAYENWGQGTQVTVSS<13F82A/1-130, SEQ ID NO: 2271; PRT; ->EVQLVESGGGLVQAGGSLRLSCAISGRTLSGRAMGWFRQAPGKEREFREFVAATSWSGGSKYVADSVTGRFTIFRDNAENTAYLQMNSLNPEDTAVYYCAVTKRYYSIKYYSTVEDYEYWGQGTQVTVSS<13E101/1-128, SEQ ID NO: 2272; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGRTFNNDHMGWFRQAPGTERELVAATGRRGGPTYYADSVKGRFTISRDNAESTVYLQMNSLKAEDTAVYYCAANRYYCSTYGCLSTPRQYDYWGQGTQVTVSS<22B85/1-120, SEQ ID NO: 2273; PRT; ->EVQLVESGGGLVRPGGSLRLSCATSGSDIGINAMGWYRQAPGNQRELVATITGSTGTTYADSVKGRFAISRDGAKNTVYLQMDSLKPEDTAVYYCNLRVYTGTYGGRNYWGQGTQTVSS<11B12/1-118, SEQ ID NO: 2274; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALINYAMGWFRQAPGKEREFVSAINWSGSHTDYGDSVKGRFAISRDNAKNTVYLQMHSLKPEDTAVYHCATGYSLPAFDSWGPGTQVTVSS<13G61/1-118, SEQ ID NO: 2275; PRT; ->EVQLVESGGGVVQAGGSLRLSCAPSGRTFSSYVMGWVRQAPGKAREFVAGITRNSGRTRYADSVKGRFTISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFHYFGQGTQVTVSS<14H41/1-118, SEQ ID NO: 2276; PRT; ->EVQLVESGGGLVQAGGSLRLSCAPSGRTFSSYVMGWVRQAPGKAREFVAGITRNSIRTRYADSVKGRFTISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFDYFGQTQVTVSS<11B81/1-126, SEQ ID NO: 2277; PRT; ->EVQLVESGGGLVQGGSLRLSCAASGRPVNNYIMGWFRQALGQGREFVAAINRNGATAAYADSVKGRFTISRDNAEDLLYLQMNLLKPEDTAVYYCAANSDSGFDSYSVWAAYEYWGQGTQVTVSS<11C11/1-121, SEQ ID NO: 2278; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSAYAMGWFRQAPGKERESVATIRWTGGSSSTSYADSVKGRFTISKNTAENTVYLQMNSLKPEDTAVYYCAVLLTVWDTYKYWGQGTQVTVSS<12D92/1-123, SEQ ID NO: 2279; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTYNMAWFRQAPGKEREFVAAMNWSGGSTKYAESVKGRFTISRANDNNPLYLQMNTLKPEDTAVYYCAATNRWYTGVYDLPSRYEYWGQGTQVTVSS<13E61/1-123, SEQ ID NO: 2280; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGQTFNMGWFRQAPGKEREFVAAISWSQYNTKYADSVKGRFTISRDNAINSLYLQMDTLKPEDTAVYYCAATNRWFSAVYDLPSRYTYWGQGTQVTVSS<22B71/1-114, SEQ ID NO: 2281; PRT; ->EVQLVESGGAFVQGGSLRLSCAASGSDVWFNVMGWYRQGPGQQLELVASITYGGNINYGDPVKGRFSISRDNALKTVYLQMNSLKPEDTAVYYCYADLPSRLWGQGTQVTVSS<21A121/1-123, SEQ ID NO: 2282; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGRAFNMGWFRQAPGKEREFVAGVNWGGGSTKVADSVKERFTISRDYDNSPVYLQMNTLKPEDTAVYYCAATSRWYSAVYDLPTRYDYWGQGTQVTVSS<13F101/1-124, SEQ ID NO: 2283; PRT; ->EVQLVESGGGLVQAGGSLRLSCQLSGGTVSDLHMGWFRQAPGKEREFVGFTRWPSITYIAEHVKGRFTISRDNAKNTVYLQMNSLEREDTAVYYCAADRSYSIDYRHADSYSYWGQGTQVTVSS<11A43/1-123, SEQ ID NO: 2284; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSIFRVNHMGWYRQAPGKQREFVAAITSDHITWYADAVKGRFTISRDNAHNTVTLQMNSLRPEDTAVYYCAADPLLFYGVGSADVDYWGQGTQVTVSS<12C81/1-117, SEQ ID NO: 2285; PRT; ->EVQLVESGGGLVQPGGSLRLSCAGSGNIVRDNTMAWYRQAPGNQRDLVATINVGGGTYYAGPVKGRFTISRDNAKNSVYLQMNSLKPEDTSVYYCNVISGLVQRDYWGQGTQVTVSS<11B21/1-124, SEQ ID NO: 2286; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSMYLMGWFRQAPGKEREFVSTINRRGGNTYYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAVYYCAAGGHLLGYDVQWEPDYWGQGTQVTVSS<11B71/1-126, SEQ ID NO: 2287; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFERYAMGWFRQAPGKEREFVATISWSGGRDTVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHKRTYELGAHSTDFGSWGQGTQVTVSS<12C121/1-126, SEQ ID NO: 2288; PRT; ->EVQLVESGGDLVQPGESLRLSCAVSGVTVDYSGIGWFRQAPEKEREAVSCIESGDCTTTYVDSVKGRFTISRDNAKNAVYLQMNSLKPEDTGVYYCATAVFVDSGDFSVCRGVGYWGKGTQVTVSS<22C51/1-121, SEQ ID NO: 2289; PRT; ->EVQLVESGGGLVQAGASLRLSCAASGRTFSRYDIGWFRQAPGKGREFVAAINWSGGTTSFGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAALRSWPRGVDSGSWGQGTQVTVSS<12D11/1-123, SEQ ID NO: 2290; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGRTFSGSRMGWFRQAPGKEREFVAAIRWSGGITWYAESVKSRFTISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQVTVSS<12D14/1-123, SEQ ID NO: 2291; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSGSRMGWLRQAPGKEREFVAAVRWSGGITWYAESVKGRFTISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQTVSS<12C111/1-123, SEQ ID NO: 2292; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGLTFSSYAMGWFRQAPGKVREFVATISRSGGRTSYADSVKGRFIVSRDNAKNTADLQMNDLKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTVSS<22B55/1-123, SEQ ID NO: 2293; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGLTFSTYAMGWFRQAPGKVREFVATISRSGGRTSYADSVKGRFIVSRDNAKNTADLQMNELKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTVSS<14H121/1-113, SEQ ID NO: 2294; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQGPGKRRELVARIAGGSTNYADSVKGRFTISRDDAKNTVFLQMNSLKPEDTAVYYCNVDGPFGNWGQGTQVTVSS<12C71/1-125, SEQ ID NO: 2295; PRT; ->EVQLVESGGGLNQAGGSLRLSCTASGGTFGSYALGWFRQSPGKERESVAAIDWDGSRTQYADSVKGRFTISRENVKDTMYLQMNSLQAEDTGVYYCVRSRHSGNTLSFSLKYDYWGQGTQVTVSS<21A31/1-125, SEQ ID NO: 2296; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASEPTFSSVAMGWFRQGPGKEREFAATITWSGDSTYVTDSVKGRFTISRDNARNTAYLQMDSLRPEDTAVYSCAARRWSGTLSLFDNEYYYWGQGTQVTVSS<12C91/1-121, SEQ ID NO: 2297; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTSSYYHMAWFRQAPGKEREFIAAINLSSGSTYYPDSVKGRFTISRGNAKNTVNLQMNSLKPEDTAVYYCAADNYRDSYLEYDYWGQGTQVTVSS<14H91/1-125, SEQ ID NO: 2298; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGRTFSNYRMAWFRQAPRKEREFVAAISRSGESTYFADSMKGRFTISRDNTESTGYLQMNNLKPEDTAVYYCAASWDHGDYVDGGFFYDYWGQGTQVTVSS<12C42/1-124, SEQ ID NO: 2299; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMHWFRQAPGSERDFVAGISWDGGSTFYANSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAAAGSAGPPSIDRQYDYWGQGTQVTVSS<12D102/1-118, SEQ ID NO: 2300; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSSLSFNAMGWSREAPGKRRELVARIISDDSTLYADSVKGRFTISRDYAKNTAYLQMNSLKPEDTAVYYCVADVRDSIWRSYWGQGTQVTVSS<11A52/1-120, SEQ ID NO: 2301; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALSNYAMRWFRQAPGKEREFVATINWSGSHTDYADSVKGRFTISRDNAENTVYLQMNSLTPEDTAVYYCASGWGATQAQSGFWGQGTQVTVSS<14H111/1-120, SEQ ID NO: 2302; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALISFAMRWFRQAPGKEREFVAAINWSGTHTDYADSVKGRFTISRDNAENTVYLLMNSLIPEDTAVYYCATGWGATQAQHGFWGQGTQVTVSS<11B61/1-120, SEQ ID NO: 2303; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTSSGYGMGWFRQAPGKEREFVAAVGWYGSTYFADSVKGRFTIYRDNAQNTMYLQMNSLKPEDTAVYYCAASSSLATISQPSSWGQGTQVTVSS<12E42/1-118, SEQ ID NO: 2304; PRT; ->EVQLVESGGGLVQPGGSLRLSCAHSGRAFSLRTMGWYRQAPGNQRELVALISAGDSTYYPDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAKAVTSRDHEYWGQGTQVTVSS<13F81A/1-128, SEQ ID NO: 2305; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAAISWTGGSSYYGDSVKGRSTISRENAENTVYLQMNSLKPEDTAVYYCAANSDEFYSGTLKLQSRMVEYWGQGTQVTVSS <11B102/1-118, SEQ ID NO: 2306; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGIFSSHAISWFRQAPGKAREFVAAINWSGSHRDYADSAKGRFTISRDNAKKTAYLQMNSLRPEDTAVYYCVGGWKTDEYVKWGQGTQVTVSS<21A41/1-120, SEQ ID NO: 2307; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRIFSNYAWSWFRQAPGKERGFVAAINWSGGYTDYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCRPGWVTPSYEYGNWGQGTQVTVSS<14H101/1-128, SEQ ID NO: 2308; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFISSPMGWFRQAPGKEREVVAATTRSGGLPYYSDSVKGRFTISRDNAKNTVDLQMSSLKPEDTAAYYCAADQKYGMSYSRLWLVSEYEYWGQGTQVTVSS<12E21/1-115, SEQ ID NO: 2309; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSIDSIHVVGWYRKAPGKQREVVAYIGTAGATHYADSVKGRFTISRDNAENLVYLQMNNLKPEDTAVYYCSAGWGDSAYWGQGTQVTVSS<13F21/1-123, SEQ ID NO: 2310; PRT; ->EVQLVESGGGLVQSGGSLRLSCVASGTIVSINATSWYRQAPGNQRELVATIIGDGRTHYADSVKDRFTISRDAAANLVYLQMNSLKPSDTAIYSCNANGIESYGWGNRHFNYWTVGTQVTVSS<12E33/1-119, SEQ ID NO: 2311; PRT; ->EVQLVESGGGMVQAGGSLRLSCAASGLTLSNYGMGWFRQAPGKEREFVSSINWSGTHTYDADFVKGRFIISRDNAKNTVYLQINSLKPEDTAVYYCAAGGWGTGRYNYWGQGTQVTVSS<13G11/1-122, SEQ ID NO: 2312; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFISNYAMGWFRQAPGKEREFVATINWSGSHSDYADSVKGRFTISRDNAKNTVYLQMNNLKSEDTAVYYCAPGWGTAPLSTSVYWGQGTQVTVSS

TABLE B-3 Nanobodies against HER2 obtained as described in Example 4<Name, SEQ ID #; PRT (protein); -> <118N121_A1_4_OK/1-127, SEQ ID NO:2113; PRT; -> EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRFTISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSS <118N121_A6_2_OK/1-123, SEQID NO: 2313; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTFSGYSVGWFRQSPGKEREFVGGINWSGRTYYVDSVKGRFTFSRDNAKNTVYLQMNSLKPEDTAIYLCAVDRFNTIANLPGEYDYWGQGTQVTVSS <118N121_B8_1_OK/1-135, SEQ ID NO:2314; PRT; -> EVQLVESGGGLVQDGGSLRLSCAASGQLANFASYAMGWFRQAPGKAREFVAAIRGSGGSTYIADPARSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCACETFNSISNLPGEYDYWGQGTQVTVSS <118N121_A2_2_OK/1-124, SEQ ID NO: 2315; PRT; ->KVQLVESGGGLVQAGGSLRLSCAASGRTFSNYSVGWFRQAPGKEREFVAALSKDGARTYYAASVKGRFTIYRDNAKNVVYLQMSVLNGEDTAVYYCAADHFTFMSNLPSEYDYWGQGTQVTVSS<118N121_A8_2_OK/1-124, SEQ ID NO: 2316; PRT; ->EVQLVESGGGLVQAGGSLTLSCVISGLTLESHAMGWFRQAPGEEREFVATIRWSGSATFYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKIYRSLSYYGDYDSWGQGTQVTVSS<118N121_B3_1_OK/1-123, SEQ ID NO: 2317; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSDLALGWFRRAPGKEREHVAAISSSGVTTIYADSVRGRFTISRDEAKNTVYLEMNSLKTDDTAVYYCAARLTMATPNQSQYYYWGQGTQVTVSS<118N121_A5_2_OK/1-114, SEQ ID NO: 2318; PRT; ->EVQLVESGGGSVQPGGSLRLSCVASGSISSTNAMGWHRQVSGKERELVAIVTDGFTNYADFAKGRFTISRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTDNWGQGIEVTVSS<118N121_A9_2_OK/1-114, SEQ ID NO: 2319; PRT; ->EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAMGWHRQVPGKQRELVAIVTDGFTNYADFAKGRFTISRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTDNWGQGIEVTVSS<118N121_A7_1_OK/1-122, SEQ ID NO: 2320; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVMGWHRQAPGKERELVSDISFGGNTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYADILYKTDITYYRNDFWGQGTQVTVSS<118N121_A10_1_OK/1-131, SEQ ID NO: 2321; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFSFADYAIGWFRQAPGKEREGVSCIANSEGTKYYADSAQGRLPISSDNAKKTVYLQMDSLKPEDTAVYYCAALPYTICPVVVKKGAVYYGVDDYWGKGTQVTVSS<118N121_A11_1_OK/1-120, SEQ ID NO: 2322; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFPFGMYGMRWVRQAPGKGPERVSSINSDGDTTYYADSVKGRFTISRDNDENMLYLQMNSLKPEDTAVYYCATGFSDRSFAVTHKGQGTQVTVSS<118N121_B7_4_OK/1-124, SEQ ID NO: 2323; PRT; ->EVQLVESGGGLEQAGGSLRLSCAASGLTFRSAAMGWFRQGPGKEREFVAAISRDGAATYYTDSVKGRFTISRDNAKNTVFLQMNSLKPEDTAIYYCAADFRLARLRVADDYDYWGQGTQVTVSS<118N121_B2_1_OK/1-130, SEQ ID NO: 2324; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFSLDDRAIAWFRQAPGKAREGVSCITPHHGGIIFTRESVKGRFATSSDSAKNTVYLQMHSLKPEDTAVYYCATLRTDYSINWANCQRDSLYGYWGQGTQVTVSS<118N121_B7_1_OK/1-119, SEQ ID NO: 2325; PRT; ->EMQLVESGGGLVQPGGSLRLSCAASGNIPPINAMAWYRQAPGNERELVAAVTSGGGTNYATSVKGRFIISRDDSKNTVDLQMNSLKPEDTAVYYCNLGGWTRTHPFDYWGQGTQVTVSS

TABLE B-4 Bivalent Nanobodies against HER2 as described in Example 12<Name, SEQ ID #; PRT (protein); -> <2A4-9GS-2A4, SEQ ID NO: 2326; PRT;-> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS <2A5-9GS-2A5, SEQ IDNO: 2327; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQG TQVTVSS <2C3-9GS-2C3,SEQ ID NO: 2328; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS <2D3-9GS-2D3, SEQ ID NO:2329; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQG TQVTVSS <5F7-9GS-5F7,SEQ ID NO: 2330; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS

TABLE B-5 Bispecific Nanobodies against HER2 and against serum albuminas described in Example 12 <Name, SEQ ID #; PRT (protein); -><2C3-9GS-ALB1, SEQ ID NO: 2331; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2A4-9GS-ALB1, SEQ ID NO:2332; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2A5-9GS-ALB1, SEQ ID NO:2333; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <2D3-9GS-ALB1, SEQ IDNO: 2334; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS <5F7-9GS-ALB1, SEQ IDNO: 2335; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS

TABLE B-6 Biparatopic Nanobodies against HER2 <Name, SEQ ID #; PRT(protein); -> <27B3-35GS-2D3, SEQ ID NO: 2336; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C3-35GS-2D3,SEQ ID NO: 2337; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27E5-35GS-2D3, SEQ ID NO: 2338; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27F2-35GS-2D3,SEQ ID NO: 2339; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D6-35GS-2D3, SEQID NO: 2340; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <30D10-35GS-2D3, SEQID NO: 2341; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <47D5-35GS-2D3, SEQID NO: 2342; PRT; ->KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<DUMMY-35GS-2D3, SEQ ID NO: 2343; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVNGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<2D3-35GS-2D3, SEQ ID NO: 2344; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<DUMMY-35GS-47D5, SEQ ID NO: 2345; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSYPMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSCAAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS<5F7-35GS-47D5, SEQ ID NO: 2346; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <47D5-35GS-5F7, SEQID NO: 2347; PRT; ->KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS <2D3-35GS-47D5, SEQID NO: 2348; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS <27F7-35GS-2D3,SEQ ID NO: 2349; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28F6-35GS-2D3, SEQID NO: 2350; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28G3-35GS-2D3, SEQ IDNO: 2351; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <28G5-35GS-2D3, SEQ IDNO: 2352; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGSGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29D9-35GS-2D3, SEQID NO: 2353; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29E9-35GS-2D3, SEQID NO: 2354; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <30E10-35GS-2D3, SEQID NO: 2355; PRT; ->KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<31D11-35GS-2D3, SEQ ID NO: 2356; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGSGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G2-35GS-2D3, SEQ IDNO: 2357; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<P27G4-35GS-2D3, SEQ ID NO: 2358; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G5-35GS-2D3, SEQID NO: 2359; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27G7-35GS-2D3, SEQID NO: 2360; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H1-35GS-2D3, SEQID NO: 2361; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H2-35GS-2D3, SEQID NO: 2362; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H3-35GS-2D3, SEQID NO: 2363; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27H4-35GS-2D3, SEQ ID NO: 2364; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27H5-35GS-2D3,SEQ ID NO: 2365; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLNESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDACTTWFEKSGSAGQGTQVTVSS<27H7-35GS-2D3, SEQ ID NO: 2366; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A3-35GS-2D3, SEQID NO: 2367; PRT; ->EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A4-35GS-2D3, SEQID NO: 2368; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27A5-35GS-2D3, SEQID NO: 2369; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B1-35GS-2D3, SEQ IDNO: 2370; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27B2-35GS-2D3, SEQ ID NO: 2371; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27B5-35GS-2D3, SEQ IDNO: 2372; PRT; ->EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQTVSS <27B7-35GS-2D3, SEQ IDNO: 2373; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27C2-35GS-2D3, SEQ ID NO: 2374; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27C5-35GS-2D3, SEQ ID NO: 2375; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27C7-35GS-2D3, SEQ IDNO: 2376; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D1-35GS-2D3, SEQID NO: 2377; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGIHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D2-35GS-2D3,SEQ ID NO: 2378; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D3-35GS-2D3, SEQID NO: 2379; PRT; ->EVQLMESGGGLVQFGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27D4-35GS-2D3, SEQID NO: 2380; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27D7-35GS-2D3, SEQ ID NO: 2381; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E2-35GS-2D3,SEQ ID NO: 2382; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E4-35GS-2D3,SEQ ID NO: 2383; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <27E7-35GS-2D3,SEQ ID NO: 2384; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29H1-35GS-2D3,SEQ ID NO: 2385; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<30H9-35GS-2D3, SEQ ID NO: 2386; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<39C1-35GS-2D3, SEQ ID NO: 2387; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS<27G8-35GS-2D3, SEQ ID NO: 2388; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS <29H2-35GS-2D3,SEQ ID NO: 2389; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQTVSS <38C6-35GS-2D3,SEQ ID NO: 2390; PRT; ->EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS

TABLE C-1 Sequence of HER2 binding Nanobodies aligned by familyHERCEPTIN ® COMPETING 13D11EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS 2B4EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS 2G2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS13D2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKIVPTDRGGHGTQVTVSS 2D5EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS 2F4EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRRGHGTQVTVSS 2C3EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS17E3 EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS17H3EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS17D2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS 2F1EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKELEWISSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS 2E2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2C2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS 2E3EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS13B10EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS 2D1EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKILPTNRGSHGTQVTVSS 2H3EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2H1EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVRGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2C1EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS15C5EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2B3EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS29H2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS17E4EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS17A2EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKVMPTDRGTHGTQVTVSS15D1EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS17B8EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS15C11EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS15G8EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKILPAERRGHGTQVTVSS17H4EVQLVESGGGLVQPGGSLRLSCVASGFSLIMYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS27G8EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS38C6EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS 2A4EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS15G7EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS15B7EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS 5G4EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS13B2EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHKDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 2E5EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS15G1EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS27B1EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS17E7EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS17D8EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 5F8EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYALSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 2D4EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS13D8EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS17G8EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2H4EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2F3EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2F5EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS30E10KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS29H1EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS17E2EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2B1EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS 2A5EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS13C12EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS17E10EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS27D4EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS15F9EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS30H9EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS39C1EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS27G2EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2D3EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 5F7EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSPBMP118N121_A1_4_OK/1-127EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAWFRQSPGKERDLNAGIMWDGRSLFYADSVKGRFTISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSSOT-FPB COMPETING 47D5KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSHER2 BINDING 14B11EVQLNESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS14B10EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS14B4EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS14C11EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS14B5EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS14C6EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS14A4EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS14B3EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS14C1EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS14A12EVQLNKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS14A2EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS14A1EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS17C3EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS46D3KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRFTVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS27H5EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSS17C2EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQGTQVTVSS17D11EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREFVATISRGGSATYYADLKKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSSLYTSSNVFEDYWGQGTQVTVSS15A6EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS17B6EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS17C5EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS15E11EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDWVATISARGMPAYEDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS15C2EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS2A3 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDWVATIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYYLCRDVNGDIWQGTQVTVSS 27A5EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS  2C5EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDWVATIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS 27G5EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS13A9EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS29E9EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS15D8EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS15G4EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS15D12EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQRELVATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS15E12EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS13D7EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS13A8 EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 15A4EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 17F7EVQLVESGGGLVQAGGSLRLSCVASGIAQS  IRVMAWYRQPPGKQRDWVGTISSDGTANYADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS 15C8EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 17A10EVQLVESGGGLVQAGGSLRLSCVASGIPSI   RAIAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 27D3EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 13B12EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATIGSDGTTIYADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 15B2EVQLVESGGGLVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS15B11EVQLVESGGGSVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS13C9EVQLVESGGGLVQAGGSLRLSCVASGIPSI  HAMAWYRQAPGKQRDWGATTYSRGG  TTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS17D5 EVQLVESGGGLVQPGGSLRLSCAASGIIGTIRTMAWYRQAPGKQRDWVA  SIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS27B5 EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRETVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS27C7 EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS13D4 EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS 15G5EVQLVESGGGLVQAGGSLRLSCVVSGIPST IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS 13C4EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS 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  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS17A8EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS15G10EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS27A3EVQLVESGGGLVQAGGSLSLSCVASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS17H10EVQLVESGGGLVQAGGSLSLSCSASGRFFS  TRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS30D10EVQLVESGGGLVQAGGSLTLSCTASETTVR  IRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS15H4EVQLVESGGGLVQAGGSLTLSCAPSESTVS  FNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS17B7EVQLVESGGGLVQAGGSLRLSCAASGIISS  FRTMAWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYWGQGTQVTVSS15D2EVQLVESGGGLVQAGGSLRLSCVVSGVFGP  IRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS17G5EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS15B6EVQLVESGGGLVQPGGSLRLSCAASGIIGS  FRTMAWYRQAPGNQRDWVATIGSRGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS27F2EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS17F5EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS17B2EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS27H4EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS13A4EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS 2A1EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFTISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS15E10EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKERELVAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS27E7EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS47E5EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS 2G4EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFTISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS14D4EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS17A5EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS15D10EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS13C2EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS17G11EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS17A3EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTISRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS27B7EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS17A6EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS17D7EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS46D4EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRFTISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS27B3EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS27E5EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS27D6EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS30D10EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS47G11EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVAAIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS27C3EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE C-2 k_(off) rate of different Nanobodies as measured in Biacore IDk_(off) (s⁻¹) 2A4 2.05E−03 2A5 1.42E−03 2A6 1.65E−03 2B1 1.55E−03 2C41.26E−03 2D2 1.61E−03 2D4 1.65E−03 2F2 1.65E−03 2F3 1.53E−03 2F51.57E−03 2G5 1.56E−03 2H4 1.61E−03 2B2 1.19E−03 2B3 1.25E−03 2B42.77E−03 2B5 1.15E−03 2C1 1.18E−03 2C2 4.12E−03 2C3 1.11E−03 2D11.27E−03 2D5 1.20E−03 2F1 1.77E−03 2F4 1.07E−03 2G1 1.23E−03 2G21.30E−03 2G3 1.20E−03 2H1 1.09E−03 2H2 1.18E−03 2H3 1.15E−03 2H51.21E−03

TABLE C-3 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values forbinding of Nanobodies 2D3, 5F7 and 47D5 to HER2. Fusion of a dummyNanobody at the N-terminal end of the Nanobodies 2D3 and 47D5 does notsignificantly impact on the binding characteristics of 2D3 or 47D5respectively. Nanobody ID k_(off) (s⁻¹) K_(on) (1/Ms) K_(D) (nM) 2D31.48E−03 1.36E+06 1.09 Dummy-2D3 1.13E−03 1.16E+06 1.77 5F7 3.02E−041.02E+06 0.29 47D5 8.62E−04 3.86E+05 2.23 Dummy-47D5 8.69E−04 2.71E+053.21

TABLE C-4 Off-rate analysis of HER2-ECD on 2D3, 47D5 and 2D3-35GS-47D5coated sensor chips Analyte Protein on sensor chip k_(off) (1/s)  100 nMHer2 ECD 2D3-47D5 8.07E−5  100 nM Her2 ECD 2D3 2.10E−3  100 nM Her2 ECD5F7 2.56E−3 1000 nM Her2 ECD 2D3-47D5 5.45E−5 1000 nM Her2 ECD 2D31.51E−3 1000 nM Her2 ECD 5F7 1.31E−3

TABLE C-5 Oligonucleotide primers used for generation of Omnitarg lightchain V_(L) + C_(L) by overlap extension For-sequences SEQ ID NORev-sequences SEQ ID NO >For_LCrescuepAX51 2396 >Rev_LC1pAX51 2408tgattacgccaagct TAATAACAATCCAGCGGCTGCCGTAG GCAATAGGTATTTCATGTTGAAAATCT >For_LC1pAX51 2397 >Rev_LC2_OT 2409 tgattacgccaagcttgcatgcaATCGCCGACGGACGCGCTCAGGCTAC aattctatttcaaggagattttcTCGGAGATTGCGTCATCTGGATGTCG aacatga GC >For_LC2pAX51_OT 2398 >Rev_LC3_OT2410 gctggattgttattactcgcggc CCGGCTTCTGTTGATACCAAGCAACCccagccggccatggccGACATCC CCGATAGATACGTCCTGACTTGCT AGATGACG >For_LC3_OT2399 >Rev_LC4_OT 2411 GCGTCCGTCGGCGATCGCGTTAC CCGCTGAAACGGGAAGGCACACCGGTCATCACATGCAAAGCAAGTCAGG GTAACGATATGATGCGGAGTAAAT ACGT >For_LC4_OT2400 >Rev_LC5_OT 2412 ATCAACAGAAGCCGGGCAAGGCT ATAGTAGGTGGCGAAGTCCTCTGGCTCCGAAATTGCTCATTTACTCCGC GCAGGCTAGAGATAGTCAGGGTAA ATCA >For_LC5_OT2401 >Rev_LC6_OT 2413 TTCCCGTTTCAGCGGAAGCGGCT TACCGTACGTTTAATTTCCACTTTCGCGGGTACTGATTTTACCCTGACT TACCCTGGCCAAAGGTATACGGGT ATCT >For_LC6_OT2402 >Rev_LCrescue_VL_OT 2414 TTCGCCACCTACTATTGTCAGCA TCGGAAGGCGGAAAGATACTATATTTACCCGTATACCT TTGG >For_LC7_OT 2403 >Rev_LC7 2415ATTAAACGTACGGTAGCTGCCCC ATACGACGCTGGCCGTACCACTTTTCTAGCGTGTTTATCTTTCCGCCTT AGCTGCTCGTCGGAAGGCGGAAAG CCGA >For_LC82404 >Rev_LC8 2416 CGGCCAGCGTCGTATGTTTACTG CCGGACTGCAGTGCATTATCCACTTTAATAACTTCTATCCGCGCGAAGC CCATTGGACTTTAGCTTCGCGCGG TAAA >For_LC92405 >Rev_LC9 2417 TGCACTGCAGTCCGGCAATTCTC GGTCAGGGTAGAGCTCAGTGAGTAAGAAGAATCCGTGACGGAACAAGAT TGCTATCTTTGCTATCTTGTTCCG AGCA >For_LC102406 >Rev_LC10 2418 AGCTCTACCCTGACCTTGTCAAA GAAAGTCCCTGATGGGTCACTTCACAGGCAGATTATGAAAAACACAAAG GGCGTAAACTTTGTGTTTT TTTA >For_LC112407 >Rev_LC11 2419 CCATCAGGGACTTTCGAGTCCGG aaatagaattggcgcgccttattaGCTTACAAAGTCTTTTAACCGCGG ACTCACCGCGGTTAAAAGAC >Rev_LCrescue 2420aaatagaattggcgc

TABLE C-6 Oligonucleotide primers used for generation of Omnitarg heavychain V_(H) + CH₁ by overlap extension SEQ ID SEQ ID For NO RevNO >For_HCrescue 2421 >Rev_HC1 2434 gtgctaataaggcgcAAAGGTACCACTAAAGGAATTGCGAA TAATAATTTTTTCACTATGACTGT >For_HC12422 >Rev_HC2_OT 2435 gtgctaataaggcgcgccaattctatACGCAGAGAACCGCCTGGCTGCACCA ttcaaggagacagtcatagtgaaaGCCCACCTCCGCTTTCCACCAGCT >For_HC2_OT 2423 >Rev_HC3_OT 2436tttagtggtacctttctattctcact TTTCACGTTCACTGATTATACCATGGccGAGGTTCAGCTGGTGGAAAGCG ATTGGGTTCGCCAGGCGCCGGGTA >For_HC3_OT2424 >Rev_HC4_OT 2437 GGCGGTTCTCTGCGTCTGAGCTGCGCCCCTTAAAACGTTGGTTGTAAATTGA TGCCTCCGGTTTCACGTTCACTGAGCCACCAGAGTTAGGGTTTACGTC >For_HC4_OT 2425 >Rev_HC5_OT 2438GCCAGGCGCCGGGTAAAGGCCTTGAA TTCTGCACGCAGCGAATTCATCTGTATGGGTGGCCGACGTAAACCCTAAC AATAGAGTGTGTTTTTAGAGCGAT >For_HC5_OT2426 >Rev_HC6_OT 2439 CCAACGTTTTAAGGGTCGTTTCACCCTGCCTTGGCCCCAATAGTCAAAGTAA TGAGCGTAGATCGCTCTAAAAACAAAGGACGGGCCCAGATTGCGTGCA >For_HC6_OT 2427 >Rev_HC7 2440TCGCTGCCTGCAGAAGACACCGCTGT GATTTCGAGCTTGGGGCCAGCGGAAATTATTACTGTGCACGCAATCTGGG CACTGACGGACCTTTAGTGCTTGC >For_HC7_OT2428 >Rev_HCrescue_VH_OT 2441 ATTGGGGCCAAGGCACGTTGGTCACC GATTTCGAGCTTGGGGTGAGTAGCGCAAGCACTAAAGGT >Rev_HC7_OT_PCR 2429 >Rev_HC8 2442ACCTTTAGTGCTTGCGCTACTCACGG GGAGACAGTGACCGGTTCCGGGAAGTTGACCAACGTGCCTTGGCCCCAAT AATCTTTCACCAGACAGCCCAGCG >For_HC8 2430 >Rev_HC92443 CCCAAGCTCGAAATCCACGTCCGGTG TATACAAGCCGCTAGACTGCAAAACCGCACCGCCGCGCTGGGCTGTCTGG GCAGGGAAAGTATGTACACCCGAG >For_HC92431 >Rev_HC10 2444 CCGGTCACTGTCTCCTGGAACTCGGGTGGTTCACATTGCAAATATACGTCTG TGCACTTACCTCGGGTGTACATACGGTGCCCAGAGAGCTTGAAGGCAC >For_HC10 2432 >Rev_HC11 2445CTAGCGGCTTGTATAGCCTGTCAAGC TTTTTGTTCTGCGGCCGCACAGCTCTGTTGTGACCGTGCCTTCAAGCTCT TCGGTTCCACTTTCTTATCCA >For_HC112433 >Rev_HCrescue 2446 TTGCAATGTGAACCACAAACCGAGTA TTTTTGTTCTGCGGCACACCAAAGTGGATAAGAAAGTGG

1. Amino acid sequence essentially consisting of a domain antibody, of asingle domain antibody, of a “dAb” or of a Nanobody®, that is directedagainst and/or that can specifically bind to HER2 and/or wherein saidamino acid sequence inhibits and/or blocks binding of Herceptin® toHER2. 2.-43. (canceled)
 44. Amino acid sequence according to claim 1,that essentially consists of a Nanobody® that i) has at least 80% aminoacid identity with at least one of the amino acid sequences of SEQ IDNO's: 1 to 22 or 2051-2325, in which for the purposes of determining thedegree of amino acid identity, the amino acid residues that form the CDRsequences are disregarded; and in which: ii) preferably one or more ofthe amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103,104 and 108 according to the Kabat numbering are chosen from theHallmark residues mentioned in Table A-3.
 45. (canceled)
 46. Amino acidsequence according to claim 1, that essentially consists of a partiallyhumanized or fully humanized Nanobody®. 47.-77. (canceled)
 78. Aminoacid sequence according to claim 1, that essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 ischosen from the group consisting of: a) the amino acid sequences of SEQID NO's: 401-463; b) amino acid sequences that have at least 80% aminoacid identity with at least one of the amino acid sequences of SEQ IDNO's: 401-463; c) amino acid sequences that have 3, 2, or 1 amino aciddifference with at least one of the amino acid sequences of SEQ ID NO's:401-463 and/or CDR2 is chosen from the group consisting of: d) the aminoacid sequences of SEQ ID NO's: 951-1013; e) amino acid sequences thathave at least 80% amino acid identity with at least one of the aminoacid sequences of SEQ ID NO's: 951-1013; f) amino acid sequences thathave 3, 2, or 1 amino acid difference with at least one of the aminoacid sequences of SEQ ID NO's: 951-1013; and/or CDR3 is chosen from thegroup consisting of: g) the amino acid sequences of SEQ ID NO's:1501-1563; h) amino acid sequences that have at least 80% amino acididentity with at least one of the amino acid sequences of SEQ ID NO's:1501-1563; i) amino acid sequences that have 3, 2, or 1 amino aciddifference with at least one of the amino acid sequences of SEQ ID NO's:1501-1563.
 79. (canceled)
 80. Amino acid sequence according to claim 78,that is a Nanobody chosen from the group consisting of SEQ ID NO's:2051-2113 or from the group consisting of amino acid sequences that havemore than 80% preferably more than 90% amino acid identity, morepreferably more than 95%, such as 99% or more sequence identity with atleast one of the amino acid sequences of SEQ ID NO's: 2051-2113. 81.Amino acid sequence that cross-blocks the binding of at least one of theamino acid sequences according to claim 78 to HER2 and/or that iscross-blocked from binding to HER2 by at least one of the amino acidsequences according to claim
 78. 82.-150. (canceled)
 151. Compound orconstruct, that comprises or essentially consists of one or more aminoacid sequences according to claim 1, and optionally further comprisesone or more other groups, residues, moieties or binding units,optionally linked via one or more linkers. 152.-160. (canceled) 161.Compound or construct according to claim 151, which is a multiparatopicconstruct, such as a biparatopic or triparatopic construct, comprisingat least one amino acid sequence directed against a first antigenicdeterminant, epitope, part or domain of HER-2 and at least one aminoacid sequence directed against a second antigenic determinant, epitope,part or domain of HER-2 different from the first antigenic determinant,epitope, part or domain. 162.-179. (canceled)
 180. Compound or constructaccording to claim 151, wherein said compound or construct is directedagainst and/or specifically binds the Herceptin® binding site on HER2and is directed against and/or specifically binds the Omnitarg bindingsite on HER2.
 181. (canceled)
 182. Compound or construct according toclaim 151, wherein said compound or construct competes with Herceptin®and Omnitarg for binding to HER2 and/or wherein said compound orconstruct inhibits and/or blocks binding of Herceptin® and Omnitarg toHER2. 183.-185. (canceled)
 186. Biparatopic compound or constructaccording to claim 161, which can simultaneously bind the Omnitargbinding site on HER2 and to the Herceptin® binding site on HER2.187.-203. (canceled)
 204. Compound or construct according to claim 151,that comprises or that is chosen from the group consisting of SEQ IDNO's: 2336-2344 and 2346-2390 or from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more sequence identity (asdefined herein) with at least one of the amino acid sequences of SEQ IDNO's: 2336-2344 and 2346-2390.
 205. (canceled)
 206. Compound orconstruct according to claim 151, which has an increased half-life,compared to the corresponding one or more amino acid sequences per se.207.-234. (canceled)
 235. Monovalent construct, comprising oressentially consisting of one amino acid sequence according to claim 1.236.-239. (canceled)
 240. Method for preparing a multivalent constructcomprising two or more binding units comprising the use of a monovalentconstruct according to claim 235, in preparing a multiparatopicconstruct such as a biparatopic construct, wherein the monovalentconstruct is used as a binding domain or binding unit in preparing themultivalent construct comprising two or more binding units. 241.-249.(canceled)
 250. Method according to claim 240 comprising use of twomonovalent constructs, wherein a first monovalent construct is directedagainst the Omnitarg binding site on HER2 and/or is capable of competingwith Omnitarg for binding to HER-2 and wherein the second monovalentconstruct is directed against the Herceptin® binding site on HER2 and/oris capable of competing with Herceptin® for binding to HER-2. 251.Nucleic acid or nucleotide sequence, that encodes an amino acid sequenceaccording to claim
 1. 252. (canceled)
 253. Method for preparing agenetic construct that encodes a multiparatopic (such as a biparatopic)construct comprising use of a nucleic acid or nucleotide sequenceaccording to claim 251, that encodes a monovalent construct. 254.(canceled)
 255. Host or host cell that expresses, or that under suitablecircumstances is capable of expressing, an amino acid sequence accordingto claim
 1. 256. Method for producing an amino acid sequence, saidmethod at least comprising the steps of: a) expressing, in a suitablehost cell or host organism or in another suitable expression system, anucleic acid or nucleotide sequence according to claim 251 optionallyfollowed by: b) isolating and/or purifying the amino acid sequence thusobtained.
 257. Method for producing an amino acid sequence, said methodat least comprising the steps of: a) cultivating and/or maintaining ahost or host cell according to claim 255 under conditions that are suchthat said host or host cell expresses and/or produces at least one aminoacid sequence, optionally followed by: b) isolating and/or purifying theamino acid sequence thus obtained.
 258. Method for preparing and/orgenerating a multiparatopic (such as e.g. biparatopic, triparatopic,etc.) construct, said method comprising at least the steps of: a)providing a nucleic acid sequence according to claim 251, encoding afirst HER2 binding amino acid sequence, fused to a set, collection orlibrary of nucleic acid sequences encoding amino acid sequences; b)screening said set, collection or library of nucleic acid sequences fornucleic acid sequences that encode a second amino acid sequence that canbind to and/or has affinity for an antigenic determinant on HER2different from the antigenic determinant recognized by the first HER2binding amino acid sequence; and c) isolating the nucleic acid sequenceencoding an HER2 binding amino acid sequence fused to the nucleic acidsequence obtained in b), followed by expressing the encoded construct;wherein the first amino acid sequence used in step a) is such that (i)it can bind to and/or has affinity for the Herceptin® binding site onHER2 and/or (ii) competes with Herceptin® for binding to HER-2.259.-263. (canceled)
 264. Method according to claim 258, wherein in stepb), the set, collection or library of nucleic acid sequences is screenedfor nucleic acid sequences that encode (i) an amino acid sequence thatcan bind to and/or has affinity for the Omnitarg binding site on HER2and/or (ii) an amino acid sequence that can compete with Omnitarg orOmnitarg Fab for binding to HER-2. 265.-270. (canceled)
 271. Method forscreening for suitable and/or optimal linker lengths for linking a firstand a second amino acid sequence in a biparatopic construct according toclaim 161, wherein said method comprises at least the steps of: a)providing a set, collection or library of nucleic acid sequences, inwhich each nucleic acid sequence in said set, collection or libraryencodes a fusion protein that comprises a first amino acid sequence thatcan bind to and/or has affinity for a first antigenic determinant, part,domain or epitope on HER2 that is fused via a linker sequence to asecond amino acid sequence that has can bind to and/or has affinity fora second antigenic determinant, part, domain or epitope on HER2 (whichmay be the same or different as the first antigenic determinant, part,domain or epitope on HER2), in which essentially each nucleic acidsequence (or most of these) encodes a fusion protein with a differentlinker sequence so as to provide a set, collection or library encodingdifferent fusion proteins; b) screening said set, collection or libraryof nucleic acid sequences for nucleic acid sequences that encode anamino acid sequence that can bind to and/or has affinity for the firstand second antigenic determinant, part, domain or epitope on HER2; andc) isolating the nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for the first and secondantigenic determinant, part, domain or epitope on HER2, optionallyfollowed by expressing the encoded amino acid sequence; wherein thesecond amino acid sequence is an amino acid sequence that can bind toand/or has affinity for the Herceptin® binding site on HER2 and/or thatcan compete with Herceptin® for binding to HER-2.
 272. Method accordingto claim 271, wherein the first amino acid sequence is an amino acidsequence that can bind to and/or has affinity for the Omnitarg bindingsite on HER2 and/or that can compete with Omnitarg or Omnitarg Fab forbinding to HER-2. 273.-286. (canceled)
 287. Method for preparing and/orgenerating a biparatopic constructs, said method comprising at least thesteps of linking two or more monovalent amino acid sequences ormonovalent construct according to claim
 235. 288. (canceled) 289.Composition, comprising at least one amino acid sequence according toclaim
 1. 290.-291. (canceled)
 292. Method for the prevention and/ortreatment of at least one cancer and/or tumor, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of at least one amino acid sequence according to claim 1.293.-404. (canceled)